Novel inhibitors of bacterial biofilms and related methods

ABSTRACT

Certain multi-cyclic compounds and compositions thereof are useful for reducing or inhibiting the growth of bacterial biofilms and for controlling bacterial biofilm infections. Such compounds and compositions are also useful in methods for reducing or inhibiting the growth of biofilms and for controlling bacterial biofilm infections involving biofilms.

FIELD OF THE INVENTION

The present invention generally relates to compounds and compositionsuseful for reducing or inhibiting the growth of a biofilm. The presentinvention also relates to compounds useful for reducing or inhibitingthe formation of a biofilm and for controlling or treating a chronicbacterial infection involving biofilms.

BACKGROUND

Bacterial biofilms exist in natural, medical, and engineeringenvironments. The biofilms offer a selective advantage to amicroorganism to ensure its survival, or allow it a certain amount oftime to exist in a dormant state until growth conditions arise.Unfortunately, this selective advantage poses serious threats to animalhealth, especially human health.

Chronic infections involving biofilms are serious medical problemsthroughout the world. For example, biofilms are involved in 65% of humanbacterial infections. Biofilms are involved in prostatitis, biliarytract infections, urinary tract infections, cystitis, lung infections,sinus infections, ear infections, acne, rosacea, dental caries,periodontitis, nosocomial infections, open wounds, and chronic wounds.

Compounds that modify biofilm formation would have a substantial medicalimpact by treating many chronic infections, reducing catheter- andmedical device-related infections, and treating lung and ear infections.The potential market for biofilm inhibitors could be enormous given thesheer number of cases in which biofilms contribute to medical problems.The inhibitors may be used to cure, treat, or prevent a variety ofconditions, such as, but are not limited to, arterial damage, gastritis,urinary tract infections, pyelonephritis, cystitis, otitis media, otitisexterna, leprosy, tuberculosis, benign prostatic hyperplasia, chronicprostatitis, chronic lung infections of humans with cystic fibrosis,osteomyelitis, bloodstream infections, skin infections, open or chronicwound infections, cirrhosis, and any other acute or chronic infectionthat involves or possesses a biofilm.

In the United States, the market for antibiotics is greater than $10billion. The antibiotic market is fueled by the continued increase inresistance to conventional antibiotics. Approximately 70% of bacteriafound in hospitals resist at least one of the most commonly prescribedantibiotics. Because biofilms appear to reduce or prevent the efficacyof antibiotics, co-administration of biofilm inhibitors couldsignificantly boost the antibiotic market.

Using the protection of biofilms, microbes can resist antibiotics at aconcentration ranging from 1 to 1.5 thousand times higher than theamount used in conventional antibiotic therapy. During an infection,bacteria surrounded by biofilms are rarely resolved by the immunedefense mechanisms of the host. It has been proposed that in a chronicinfection, a biofilm gives bacteria a selective advantage by reducingthe penetration of an antibiotic into the depths of the tissue needed tocompletely eradicate the bacteria's existence (Costerton, J. W. et al.,Science. 1999 May 21; 284(5418):1318-22).

Traditionally, antibiotics are discovered using the susceptibility testmethods established by the Clinical Laboratory and Standards Institute(CLSI). The methods identify compounds that specifically affect growthor death of bacteria. These methods involve inoculation of a bacterialspecies into a growth medium, followed by the addition of a testcompound, and then plot of the bacterial growth over a time periodpost-incubation. Unfortunately these antibiotics derived from the CLSImethods would not be effective therapeutics against chronic infectionsinvolving biofilms because the methods do not test compounds againstbacteria in a biofilm. Consistently, numerous publications have reporteda difference in gene transcription in bacteria living in biofilms frombacteria in suspension, which further explains the failure ofconventional antibiotics to eradicate biofilm infections (Sauer, K. etal. J. Bacteriol. 2001, 183: 6579-6589).

Biofilm inhibitors can provide an alternative treatment approach forcertain infections. Biofilm inhibitors, on the other hand, act on thebiological mechanisms that provide bacteria protection from antibioticsand from a host's immune system. Biofilm inhibitors may be used to“clear the way” for the antibiotics to penetrate the affected cells anderadicate the infection. Traditionally, treatment of nosocomialinfections requires an administration of a combination of products, suchas amoxicillin/clavulanate and quinupristin/dalfopristin, or anadministration of two antibiotics simultaneously. In one study ofurinary catheters, rifampin was unable to eradicatemethicillin-resistant Staphylococcus aureus in a biofilm but waseffective against planktonic, or suspended cells (Jones, S. M., et. al.,“Effect of vancomycin and rifampicin on methicillin-resistantStaphylococcus aureus biofilms”, Lancet 357:40-41, 2001).

Bacteria have no known resistance to biofilm inhibitors. Biofilminhibitors are not likely to trigger growth-resistance mechanisms oraffect the growth of the normal human flora. Thus, biofilm inhibitorscould potentially extend the product life of antibiotics.

Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients isresistant to high doses of antibiotics because it forms biofilms.Biofilms are complex, heterogeneous communities of bacterial cellsencased in extrapolymeric substances (EPS). These EPS are composed ofpolysaccharides, proteins, and extracellular DNA. EPS provide frameworksfor communities of bacteria to exist and enhance attachment tothemselves and surfaces. Bacteria within biofilms differentiate intostratified communities of phenotypically diverse cells that offercompetitive advantages for different environmental conditions. One ofthese advantages is increased tolerance to antibiotics, which enablesbacteria like P. aeruginosa to persist in chronic infections despiteantibiotic therapy. While residing within these EPS, one hypothesis isthat P. aeruginosa reduces its metabolism, which prevents its death fromantibiotics. As antibiotic concentrations are reduced, specificpopulations of P. aeruginosa within the EPS increase their metabolismand spread. This cycle continuously repeats, enabling the spread of P.aeruginosa bacteria within the lungs of CF patients.

Chronic wound infection represents another illness that is difficult toeradicate. Examples of the most common types of chronic wounds arediabetic foot ulcers, venous leg ulcers, arterial leg ulcers, andpressure ulcers. Diabetic foot ulcers appear to be the most prevalent.These wounds are typically colonized by multiple species of bacteriaincluding Staphylococcus spp., Streptococcus spp., Pseudomonas spp. andGram-negative bacilli (Lipsky, B. Medical Treatment of Diabetic FootInfections. Clin. Infect. Dis. 2004, 39, p. S104-14).

Based on clinical evidence, microorganisms cause or contribute tochronic wound infections. Only recently have biofilms been implicated inthese infections (Harrison-Balestra, C. et al. A Wound-isolatedPseudomonas aeruginosa Grow a Biofilm In Vitro Within 10 Hours and IsVisualized by Light Microscopy, Dermatol Surg 2003, 29; 631-635;Edwards, R. et al. Bacteria and wound healing. Curr Opin Infect Dis,2004, 17; 91-96). Approximately 140,000 amputations occur each year inthe United States due to chronic wound infections that could not betreated with conventional antibiotics. Unfortunately, treating theseinfections with high doses of antibiotics over long periods of timecontributes to the development of antibiotic resistance (Howell-Jones,R. S., et al. A review of the microbiology, antibiotic usage andresistance in chronic skin wounds. J. Antimicrob. Ther. January 2005).Biofilm inhibitors in a combination therapy with antibiotics may providean effective alternative to the treatment of chronic wounds.

Recent publications describe the cycles of the pathogenesis of numerousspecies of bacteria involving biofilms. For example, Escherichia coli,which cause recurrent urinary tract infections, undergo a cycle ofbinding to and then invading a host's bladder epithelial cells. E. coliform a biofilm intracellularly, modify its morphology, and then burstout of the host cells to repeat the cycle of pathogenesis (Justice, S.et al. Differentiation and development pathways of uropathogenicEscherichia coli in urinary tract pathogenesis, PNAS 2004, 101(5):1333-1338). The authors suggest that this repetitive cycle ofpathogenesis of E. coli may explain the recurrence of the infection.

In 1997, Finlay, B. et al. reported that numerous bacteria, includingStaphylococci, Streptococci, Bordetella pertussis, Neisseria spp.,Helicobactor pylori, and Yersinia spp., adhere to mammalian cells duringtheir pathogenesis. The authors hypothesized that the adherence wouldlead to an invasion of the host cell. Later publications confirm thishypothesis (Cossart, P. Science, 2004, 304; 242-248; see additionalreferences infra). Other publications presented similar hypotheses toMulvey, M. et al. (Mulvey, M. et al. “Induction and Evasion of HostDefenses by Type 1-Piliated Uropathogenic E. coli” Science 1998, 282 p.1494-1497). In particular, Mulvey, M. et al. stated invasion of E. coliinto epithelial cells provide protection from the host's immune responseto allow a build up of a large bacterial population.

Cellular invasion and biofilm formation appear to be integral to thepathogenesis of most, if not all bacteria. P. aeruginosa have been shownto invade epithelial cells during lung infections (Leroy-Dudal, J. etal. Microbes and Infection, 2004, 6, p. 875-881). P. aeruginosa are theprincipal infectious organisms found in the lungs of cystic fibrosispatients, and the bacteria exist within a biofilm. Antibiotics liketobramcyin, and other current antibacterial compounds, do not provideeffective treatment against biofilms of chronic infections, perhapsbecause antibiotic therapy fails to eradicate the biofilm.

The pathogenesis of cellular invasion and biofilm formationgram-negative bacteria follow conserved mechanisms. For example,Haemophilus influenzae invade epithelial cells and form biofilms (Hardy,G. et al., Methods Mol. Med., 2003, 71; 1-18; Greiner, L. et al.,Infection and Immunity, 2004, 72(7); 4249-4260). Burkholderia spp.invade epithelial cells and form biofilm (Utaisincharoen, P. et al.,Microb Pathog. 2005, 38(2-3); 107-112; Schwab, U. et al. Infection andImmunity, 2003, 71(11); 6607-6609). Klebsiella pneumoniae invadeepithelial cells and form biofilm (Cortes, G et al. Infection andImmunity. 2002, 70(3); 1075-1080; Lavender, hours. et al., Infection andImmunity. 2004, 72(8); 4888-4890). Salmonella spp. invade epithelialcells and form biofilms (Cossart, P. Science, 2004, 304; 242-248;Boddicker, J. et al., Mol. Microbiol. 2002, 45(5); 1255-1265). Yersiniapestis invade epithelial cells and form biofilms (Cossart, P. Science,2004, 304; 242-248; Jarrett, C. et al. J. Infect. Dis., 2004, 190;783-792). Neisseria gonorrhea invade epithelial cells and form biofilms(Edwards, J. et al., Cellular Micro., 2002, 4(9); 585-598; Greiner, L.et al., Infection and Immunity. 2004, 73(4); 1964-1970). Burkholderiaspp. are another important class of gram-negative bacterial pathogens.Chlamydia spp., including Chlamydia pneumoniae is an intracellular,gram-negative pathogen implicated in respiratory infections and chronicdiseases such as atherosclerosis and Alzheimer's disease (Little, C. S.et al., Infection and Immunity. 2005, 73(3); 1723-34).

These Gram-negative bacteria cause lung, ear, and sinus infections,gonorrhoeae, plague, diarrhea, typhoid fever, and other infectiousdiseases. E. coli and P. aeruginosa are two of the most widely studiedGram-negative pathogens. Researchers believe that the pathogenesis ofthese bacteria involves invasion of host cells and formation ofbiofilms. These models have enabled those skilled in the art tounderstand the pathogenesis of other Gram-negative bacteria.

Accordingly, for the reasons discussed above and others, there exists anunmet need for compounds that serve as biofilm inhibitors and/or thatwould be useful for reducing or inhibiting the formation or growth ofbacterial biofilms and bacterial infections involving biofilms.

SUMMARY OF INVENTION

The present invention provides compounds of the following chemicalStructure I

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, lower cycloalkyl, lower cycloalkenyl, substituted loweralkynyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;R² is selected from the group consisting of carboxyl, amide,hydroxyamide, methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

R³ is selected from the group consisting of hydrogen or methyl; one ofR⁴ and R⁵ is hydrogen and the other is methyl; R⁶ and R⁷ areindependently selected from the group consisting of hydrogen, methyl,ethyl, propyl, isopropyl, and butyl; R⁸ selected from the groupconsisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; and wherein R⁹ isselected from the group consisting of hydrogen, halide, lower alkyl,lower alkenyl, lower alkynyl, morpholinyl, piperazinyl, lower alkylpiperazinyl, heterocycloalkyl, lower cycloalkyl, lower cycloalkenyl; andlower alkyl, lower alkenyl, and lower alkynyl substituted with moietiesselected from the group consisting of hydroxyl, amino, lower aminoalkyl,halide, lower alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, loweralkylcarbonylamino, carboxyl, amide, hydroxyamide, —CONHCH₃, —NHCONH₂,—SO₂NH₂, —SO₂CH₃, —NHCOCH₃, —NHCSNH₂, and —NHSO₂CH₃. Salts are alsocontemplated by the present invention as described in the examples.

A number of the compounds of the invention and intermediates may existin different tautomeric forms. All such tautomeric forms are within thescope of the invention. The depiction of any tautomer herein is notintended to limit the scope of the invention to one specific tautomer.For example, the following is within the scope of the invention.

Those skilled in the art will understand that Structure I may exist as atautomer envisioned as the following. In any respect, 3-aminopyrazole ofany structure of the invention may be as follows:

Or a 3-aminopyrazole of any structure of the invention may be asfollows:

These tautomeric depictions are within the scope of the invention.

Furthermore, the hydroxypyrazole of compound 68 as described herein mayexist as tautomers. These tautomers are within the scope of theinvention.

Compositions containing the compounds described above and apharmaceutically acceptable carrier are also contemplated by thisinvention. Such compositions containing the compounds described aboveoptionally include an antimicrobial agent. As demonstrated herein suchcompositions are useful in reducing or inhibiting the formation orgrowth of biofilms.

This invention also provides methods for reducing or inhibiting theformation or growth of biofilms comprising contacting the biofilm orcell capable of biofilm formation with an effective amount of acomposition or a compound of the preceeding chemical Structure I whereinR¹ is selected from the group consisting of hydrogen, methyl, halide,lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl, substitutedlower alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl,substituted lower alkynyl, lower cycloalkyl, lower cycloalkenyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl; R² is selectedfrom the group consisting of carboxyl, amide, hydroxyamide, methylamide,—CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

R³ is selected from the group consisting of hydrogen or methyl; one ofR⁴ and R⁵ is hydrogen and the other is methyl; R⁶ and R⁷ areindependently selected from the group consisting of hydrogen, methyl,ethyl, propyl, isopropyl, and butyl; R⁸ selected from the groupconsisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; and R⁹ is selectedfrom the group consisting of hydrogen, halide, lower alkyl, loweralkenyl, lower alkynyl, morpholinyl, piperazinyl, lower alkylpiperazinyl, heterocycloalkyl, lower cycloalkyl, lower cycloalkenyl; andlower alkyl, lower alkenyl, and lower alkynyl substituted with moietiesselected from the group consisting of hydroxyl, amino, lower aminoalkyl,halide, lower alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, loweralkylcarbonylamino, carboxyl, amide, hydroxyamide, —CONHCH₃, —NHCONH₂,—SO₂NH₂, —SO₂CH₃, —NHCOCH₃, —NHCSNH₂, and —NHSO₂CH₃. Salts are alsocontemplated by the present invention as described in the examples.

Inhibition or reduction of the formation or growth of biofilms may beeffected either in vivo or in vitro. Compositions used to inhibit orreduce the formation or growth of biofilms may further include anantimicrobial agent, biocide, or antibiotic. The methods also providefor inhibiting or reducing the formation or growth of biofilms on avariety of substrates.

Inhibition or reduction of the formation or growth of biofilms reducesvirulence of gram-negative bacteria. Adhesion, biofilm growth, invasion,and the secretion of enzymes or toxins contribute to virulence ofgram-negative bacteria. It is well known to those skilled in the artthat biofilms increase the virulence of gram-negative bacteria and anupregulation of virulence factors (i.e. enzymes, toxins) has beendemonstrated in gram-negative bacterial biofilms. The compounds of theinvention reduce virulence of gram-negative bacteria.

Compound 1 as described herein modulates cysB and genes under itscontrol as described in U.S. Pat. No. 7,604,978 incorporated herein inits entirety by this reference. It is anticipated that all of thecompounds described herein modulate cysB and genes under its control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of photographs of the Biofilm Growth Assay for Compound1, as described in Example I, below, wherein the round plates on theleft are negative controls, in which the agar in the plates does notcontain Compound 1, and wherein, as shown in the negative controlplates, P. aeruginosa spread out from the inoculation point in thecenter of the plate during overnight incubation at 37° C. and coveredthe entire plate, whereas the agar in the plates on the right contained0.5 μg/ml of Compound 1 of the invention, which prevented the growth ofthe spreading P. aeruginosa biofilm.

FIG. 2 is a set of photographs of the Biofilm Growth Assay for Compound1 in the presence of colistin (antibiotic) disks as described in ExampleVIII, below, wherein the top left plate was a negative control platewith paper disks without antibiotic and the top right plate had 0.125μg/ml of Compound 1 in the agar on the right side of the plate withpaper disks without antibiotic (and 0.125 μg/ml of Compound 1 was twodilutions below its optimal working concentration of 0.5 μg/ml), and thebottom left plate had colistin 10 μg disks, while the bottom right platehad 0.125 μg/ml of Compound 1 in the agar on the right side of the platewith colistin 10 μg disks, the inhibition of the growth of P. aeruginosabiofilm in the bottom right plate demonstrating that Compound 1 andcolistin (antibiotic) are synergistic at preventing the growth of P.aeruginosa biofilms.

DESCRIPTION OF THE INVENTION Definitions

“Acceptable carrier” refers to a carrier that is not deleterious to theother ingredients of the composition and is not deleterious to materialto which it is to be applied.

“Pharmaceutically acceptable carrier” refers to a carrier that is notdeleterious to the other ingredients of the composition and is notdeleterious to the human or other animal recipient thereof.“Agriculturally acceptable carrier” refers to a carrier that is notdeleterious to the other ingredients of the composition and is notdeleterious to the plant recipient thereof. In the context of the otheringredients of the composition, “not deleterious” means that the carrierwill not react with or degrade the other ingredients or otherwiseinterfere with their efficacy. Interference with the efficacy of aningredient does not encompass mere dilution of the ingredient. In thecontext of the animal or plant host, “not deleterious” means that thecarrier is not injurious or lethal to the plant or animal.

“Administration” refers to any means of providing a compound orcomposition to a subject. Non-limiting examples of administration meansinclude oral, topical, rectal, percutaneous, parenteral injection,intranasal and inhalation delivery.

“Biofilm” refers to an extracellular matrix in which microorganisms aredispersed and/or form colonies. The biofilm typically is made ofpolysaccharides and other macromolecules.

“Biofilm Growth Assay” refers to an assay performed on semi-solid agarsurface where the bacteria collectively move out from an inoculationpoint. To those skilled in the art, the act of bacteria collectivelymoving out from an inoculation point on a semi-solid surface may bereferred to as swarming or a spreading biofilm. (Anderson, et alMicrobiology, 2003, 149, 37-46; Daniels, et al. FEMS MicrobiologyReviews, 2004, 28, 261-289). The procedure to perform this assay isdescribed in detail in the Examples. This type of surface motility bybacteria has been reported in the literature by those skilled in theart. (Shrout, et al. Molecular Microbiology, 2006, 62(5), 1264-1277;Kim, et al. J. Bacteriology, 2003, 185(10), 3111-3117; Lai, et al.Environmental Microbiology, 2009, 11(1), 126-136; Overhage, et al. J.Bacteriology, 2008, 190(8), 2671-2679) These literature references alsodemonstrate a link to this type of surface motility and increasedantibiotic tolerance. These references are incorporated herein byreference in their entirety.

“Commercial source” refers to a vendor that provides the desiredcompound.

“Direct synthesis” refers to production of the desired compound byreacting appropriate compound precursors under appropriate conditions toobtain the desired compound.

“Effective amount” refers to the amount of compound or composition that,in the case of biofilm formation, will reduce the size or volume ofexisting biofilms; reduce the rate at which bacteria are capable ofproducing biofilm; or will inhibit or prevent the formation of biofilmby one or more microorganisms. In the context of treating a bacterialinfection, an “effective amount” refers the amount of a compound orcomposition that will reduce the degree of an existing infection or willinhibit or prevent an infection from occurring.

“Essentially pure preparation” refers to a preparation in which theconcentration of the desired ingredient is at least 95% or more of thepreparation by weight. In the context of this processes used in thisinvention, the antimicrobial agents and compounds of the inventiontypically and preferably make up 99% or more by weight of thepreparation and are referred to herein as “highly pure” preparations.

“In vivo”, in the context of biofilm formation, refers to effectsmediated in or upon living organisms or subjects. Effects mediated onbiofilms associated with medical devices such as central venouscatheters, urinary catheters, endotracheal tubes, mechanical heartvalves, pacemakers, vascular grafts, stents, and prosthetic jointslocated within a living organism or subject are considered as “in vivo”uses of the compounds and compositions described herein.

“In vitro”, in the context of biofilm formation, refers to effectsmediated on substrates located outside of an organism that are potentialsites of biofilm formation. Non-limiting examples of substrates includevessel hulls, cars, airplanes, industrial equipment, devices, membranes,filters, microtiter plates, continuous flow chambers, bioreactors,fermentors, chemostats and machinery.

“Is one that permits” as it relates to a pharmaceutically acceptablecarrier that has characteristics that enable the preparation to be usedfor a given mode of administration of the composition. For example,pharmaceutically acceptable carriers that permit parenteraladministration to an animal are liquids that are not injurious or lethalto the animals when so injected. Such carriers often comprise sterilewater, which may be supplemented with various solutes to increasesolubility. Sterile water or sterile water supplemented with solutes isthus a pharmaceutically acceptable carrier that permits parentaladministration.

As used herein, all agar percentages are expressed in terms ofweight/volume; all formulation percentages are expressed in terms ofweight/weight.

“Reducing or inhibiting” in reference to a biofilm refers to theinhibiting or reducing of biofilm formation or growth, a reduction inthe rate of biofilm formation or growth, reduction or removal ofpreformed or existing biofilm, as well as the partial or completeinhibition of biofilm formation or growth. This definition includes butis not limited to the biofilm growth that also occurs on semi-solidsurfaces like 0.4% to 1.0% agar, but is not limited to these surfaces,as described in the Examples of the invention. This type of surfacemotility, which is also referred to as swarming or spreading biofilmsand conducted on the same semi-solid agar plates independent ofterminology, demonstrates mechanistic relationships between attachedbiofilms and gram-negative bacteria spreading across semi-solid agarplates as known to those skilled in the art. Michael Givskov andcolleagues defined the movement of a Pseudomonas spp. across a 0.6%Bacto agar plate as surface motility with biofilm structures instead ofswarming (Anderson, et al Microbiology, 2003, 149, 37-46). Jan Michielsand colleagues defined swarming as a biofilm spreading over a semi-solidsurface (Daniels, et al. FEMS Microbiology Reviews, 2004, 28, 261-289).Even though terminology is evolving as more investigations are reported,all of these data have been generated examining the movement ofgram-negative bacteria on 0.4% to 0.7% agar surfaces.

“Subject in need thereof” refers to living organism that would benefitfrom either prevention or reductions in the degree of a bacterialinfection. Subjects may include animals or more specifically, mammals orhumans. Subjects may also include plants.

“Substrate” refers to any material to which the compound or acomposition containing the compound may be applied.

The term “lower alkyl” as used herein refers to a saturated hydrocarbonchain having one, two, three, four, or five carbon atoms. Lower alkylgroups may be optionally substituted with one or more substituents asdefined herein to form substituted lower alkyl groups. Lower alkylgroups may be straight or branched. Examples of lower alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl, and isopentyl.

The phrase “substituted lower alkyl” as used herein, refers to a loweralkyl group, as previously defined, substituted by independentreplacement of one, two, or three of the hydrogen atoms thereon withsubstituents including halide, nitrile, aryl, heteroaryl, substitutedheteroaryl, lower cycloalkyl, lower cycloalkenyl, —SH, and lowerthioalkyl.

The term “lower alkenyl” as used herein refers to an unsaturatedhydrocarbon chain having one, two, three, four, or five carbon atoms andhaving one or more carbon-carbon double bonds within the chain. Thelower alkenyl groups may be straight or branched and may be optionallysubstituted with one or more substituents as defined herein to formsubstituted lower anlkenyl groups.

The phrase “substituted lower alkenyl” as used herein, refers to a loweralkenyl group, as previously defined, substituted by independentreplacement of one, two, or three of the hydrogen atoms thereon withsubstituents including halide, nitrile, aryl, heteroaryl, substitutedheteroaryl, lower cycloalkyl, lower cycloalkenyl, —SH, and lowerthioalkyl.

The term “lower alkynyl” as used herein refers to an unsaturatedhydrocarbon chain having one, two, three, four, or five carbon atoms andhaving one or more carbon-carbon triple bonds within the chain. Thelower alkynyl groups may be straight or branched and may be optionallysubstituted with one or more substituents as defined herein to formsubstituted lower alkynyl groups.

The phrase “substituted lower alkynyl” as used herein refers to a loweralkynyl group, as previously defined, substituted by independentreplacement of one, two, or three of the hydrogen atoms thereon withsubstituents including halide, nitrile, aryl, heteroaryl, substitutedheteroaryl, lower cycloalkyl, lower cycloalkenyl, —SH, and lowerthioalkyl.

The phrase “lower alkyl ethers” as used herein refers to ethers of theformula R′OR″, wherein R′ is a lower alkyl, lower alkenyl or loweralkynyl, and R″ is a lower alkyl, lower alkenyl, lower alkynyl, or aryl,heteroaryl or heterocycloalkyl.

The term “lower alkoxy,” as used herein by itself or as part of anothersubstituent, means a radical of the formula —OR, wherein R is a loweralkyl, lower alkenyl, lower cycloalkyl, or lower cycloalkenyl group asdefined herein. Representative examples of lower alkoxy groups include,but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy,tert-butoxy, cyclopropyloxy, cyclopentyloxy, and the like.

The term “lower alkoxyalkyl” as used herein refers to a lower alkylmoiety as defined herein with one carbon atom replaced with an oxygenatom. Examples include —CH₂CH₂OCH₃ and —CH₂OCH₂CH₂CH₃.

The term “lower alkoxycarbonyl” as used herein by itself or as part ofanother substituent, refers to a radical of the formula —C(O)-(loweralkoxy), wherein lower alkoxy is as defined herein.

The phrase “lower alkyl nitrile” as used herein refers to lower alkyl orlower alkenyl with one nitrile group replacing one terminal carbon atomin the unbranched or branched chain. Lower alkyl nitrile includes, butis not limited to, —CH₂CN and —CH₂CH₂CN.

The term “lower aminoalkyl” as used herein refers to lower alkyl orlower alkenyl with one nitrogen atom replacing one carbon atom in theunbranched or branched chain. Lower aminoalkyl includes, but is notlimited to, —NHCH₂CH₂CH₃, —CH₂CH₂—NHCH₃, —CH₂N(CH₃)₂, —N(CH₃)₂, and—CH₂CH₂NH₂.

The term “lower alkylcarbonylamino” as used herein refers to lower alkylwith a carbonylamino or aminocarbonyl replacing two carbon atoms in theunbranched or branched chain. Lower alkylcarbonylamino includes, but isnot limited to, —NHCOCH₂CH₃, —CH₂CONHCH₃, —CON(CH₃)₂, and —CONHCH₃(methylamide).

The terms “halo” and “halogen,” as used herein, mean an atom selectedfrom fluorine, chlorine, bromine and iodine and the term “halide” andused herein means the corresponding anion.

The term “lower haloalkyl” as used herein refers to a lower alkyl groupwherein one or more hydrogen atoms attached to a member atom within thelower alkyl group is replaced with 1, 2, 3, or 4 halide atoms. Lowerhaloalkyl includes, but are not limited to, fluoromethyl, —CF₃,difluoroethyl, and trifluoromethyl.

The term “lower hydroxyalkyl” as used herein refers to lower alkyl orlower alkenyl wherein one or more hydrogen atoms attached to a memberatom within the lower alkyl or lower alkenyl group is replaced with oneor two hydroxyls. Lower hydroxyalkyl includes, but is not limited to,—CH₂CH₂—OH and —CH₂CH(—OH)CH₃, and the like.

The term “aryl” as used herein, means mono- or bicyclic carbocyclic ringsystems comprising 6 to 12 carbon atoms, which consist of one or twoaromatic rings, specifically including phenyl, naphthyl,tetrahydronaphthyl, indanyl, and idenyl; and specifically substitutionsto these aryls by independent replacement of one two, or three of thehydrogen atoms thereon with substituents specifically selected from thegroup consisting of halide, lower haloalkyl, amino, hydroxyl, loweralkoxy, lower aminoalkyl, lower hydroxyalkyl, nitrile, lower alkylnitrile, lower alkyl, lower alkenyl, lower alkynyl, nitro, carboxyl,amide, hydroxyamide, lower alkyl ethers, lower alkoxyalkyl, loweralkoxycarbonyl, lower alkylcarbonylamino, —SH, and lower thioalkyl.

The phrase “substituted aryl,” as used herein, means a aryl group, aspreviously defined, substituted by independent replacement or one or twoof the hydrogen atoms thereon with substituents specifically selectedfrom the group consisting of —(CH₂)₀₋₃-lower cycloalkyl, —(CH₂)₀₋₃-aryl,—(CH₂)₀₋₃-lower cycloalkenyl, —(CH₂)₀₋₃-heteroaryl,—(CH₂)₀₋₃-heterocycloalkyl, —NH-lower cycloalkyl, —NH-aryl, —NH-lowercycloalkenyl, —NH-heteroaryl, —NH-heterocycloalkyl, —O-aryl,—O-heteroaryl, —O-heterocycloalkyl, —C(O)-lower alkyl, —C(O)-loweralkenyl, —C(O)-lower alkynyl, —C(O)-lower cycloalkyl, —C(O)-aryl,—C(O)-lower cycloalkenyl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl,—CONH₂, —CONH-lower alkyl, —CONH-lower alkenyl, —CONH-lower alkynyl,—CONH-lower cycloalkyl, —CONH-aryl, —CONH-lower cycloalkenyl,—CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO₂-lower alkyl, —OCO₂-loweralkenyl, —OCO₂-lower alkynyl, —OCO₂-lower cycloalkyl, —OCO₂-aryl,—OCO₂-lower cycloalkenyl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl,—OCONH₂, —OCONH-lower alkyl, —OCONH-lower alkenyl, —OCONH-lower alkynyl,—OCONH-lower cycloalkyl, —OCONH-aryl, —OCONH-lower cycloalkenyl,—OCONH-heteroaryl, —OCONH-heterocycloalkyl, —NHC(O)-lower alkyl,—NHC(O)-lower alkenyl, —NHC(O)-lower alkynyl, —NHC(O)-lower cycloalkyl,—NHC(O)-aryl, —NHC(O)-lower cycloalkenyl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂-lower alkyl, —NHCO₂-lower alkenyl,—NHCO₂-lower alkynyl, —NHCO₂-lower cycloalkyl, —NHCO₂-aryl, —NHCO₂-lowercycloalkenyl, —NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH-lower alkyl, —NHC(O)NH-lower alkenyl, —NHC(O)NH-lower alkynyl,—NHC(O)NH-lower cycloalkyl, —NHC(O)NH-aryl, —NHC(O)NH-lowercycloalkenyl, —NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl,—NHC(S)NH₂, —NHC(S)NH-lower alkyl, —NHC(S)NH-lower alkenyl,—NHC(S)NH-lower alkynyl, —NHC(S)NH-lower cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-lower cycloalkenyl, —NHC(S)NH-heteroaryl,—NHC(S)NH-heterocycloalkyl, —S(O)-lower alkyl, —S(O)-lower alkenyl,—S(O)-lower alkynyl, —S(O)-lower cycloalkyl, —S(O)-aryl, —S(O)-lowercycloalkenyl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl, —CH₂SO₂CH₃,—SO₂NH₂, —SO₂NH-lower alkyl, —SO₂NH-lower alkenyl, —SO₂NH-lower alkynyl,—SO₂NH-lower cycloalkyl, —SO₂NH-aryl, —SO₂NH-lower cycloalkenyl,—SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl, —NHSO₂-lower alkyl,—NHSO₂-lower alkenyl, —NHSO₂-lower alkynyl, —NHSO₂-lower cycloalkyl,—NHSO₂-aryl, —NHSO₂-lower cycloalkenyl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —S-lower alkenyl, —S-lower alkynyl, —S-lowercycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl.

The phrase “lower cycloalkyl,” as used herein, means a saturatedcarbocyclic ring compound specifically including cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, norbornane, and adamantine; andspecifically substitutions to these lower cycloalkyls by independentreplacement of one or two of the hydrogen atoms thereon withsubstituents specifically selected from the group consisting of halide,lower haloalkyl, amino, hydroxyl, lower alkoxy, lower aminoalkyl, lowerhydroxyalkyl, nitrile, lower alkyl nitrile, lower alkyl, lower alkenyl,lower alkynyl, nitro, carboxyl, amide, hydroxyamide, lower alkyl ethers,lower alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, —SH,and thioalkyl.

The term “lower cycloalkenyl” as used herein specifically refers to anunsaturated hydrocarbon ring with five or six carbons; and specificallysubstitutions to these lower cycloalkenyls by independent replacement ofone or two of the hydrogen atoms thereon with substituents specificallyselected from the group consisting of halide, lower haloalkyl, amino,hydroxyl, lower alkoxy, lower aminoalkyl, lower hydroxyalkyl, nitrile,lower alkyl nitrile, lower alkyl, lower alkenyl, lower alkynyl, nitro,carboxyl, amide, hydroxyamide, lower alkyl ethers, lower alkoxyalkyl,lower alkoxycarbonyl, lower alkylcarbonylamino, —SH, and thioalkyl.

The term “heteroaryl” as used herein specifically refers to pyrrolyl,pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl,isothiazolyl, thienyl, furanyl, furazanyl, pyridinyl, pyrimidinyl,pyridazinyl, indolyl, 3H-indolyl, isoindolyl, indolinyl, indolizinyl,indazolyl, dihydroindolyl, tetrahydroindolyl, purinyl, pyrazinyl,quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, benzimidazolyl, benzopyranyl,benzoxazolyl, benzisoxazolyl, benzofuranyl, isobenzofuranyl,benzothiazolyl, benzisothiazolyl, benzothienyl, furopyridinyl,phthalazinyl, napthyridinyl, pyrazolopyridyl, pyrazolopyrimidinyl;specifically their partially reduced forms as known to those skilled inthe art like as tetrahydroisoquinolinyl is to isoquinolinyl; andspecifically a heteroaryl (as defined herein) of 6 members or less fusedwith an aryl (as defined herein) of 6 members or less OR separately twoheteroaryls (as defined herein) of 6 members or less fused together asknown to those skilled in the art like as in pyrrolopyridinyl and itspartially reduced form dihydropyrrolopyridinyl; and specificallysubstitutions to these heteroaryls by independent replacement of onetwo, or three of the hydrogen atoms thereon with substituentsspecifically selected from the group consisting of halide, lowerhaloalkyl, amino, hydroxyl, lower alkoxy, lower aminoalkyl, lowerhydroxyalkyl, nitrile, lower alkyl nitrile, lower alkyl, lower alkenyl,lower alkynyl, nitro, carboxyl, amide, hydroxyamide, lower alkyl ethers,lower alkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, —SH,and thioalkyl; and heteroaryls as defined herein with substitutions byindependent replacement of a hydrogen atom on a ring nitrogenspecifically selected from the group consisting of lower alkyl, loweralkenyl, and lower haloalkyl.

The phrase “substituted heteroaryl,” as used herein, means a heteroarylgroup as previously defined, substituted by independent replacement orone or two of the hydrogen atoms thereon with substituents specificallyselected from the group consisting of —(CH₂)₀₋₃-lower cycloalkyl,—(CH₂)₀₋₃-aryl, —(CH₂)₀₋₃-lower cycloalkenyl, —(CH₂)₀₋₃-heteroaryl,—(CH₂)₀₋₃-heterocycloalkyl, —NH-lower cycloalkyl, —NH-aryl, —NH-lowercycloalkenyl, —NH-heteroaryl, —NH-heterocycloalkyl, —O-aryl,—O-heteroaryl, —O-heterocycloalkyl, —C(O)-lower alkyl, —C(O)-loweralkenyl, —C(O)-lower alkynyl, —C(O)-lower cycloalkyl, —C(O)-aryl,—C(O)-lower cycloalkenyl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl,—CONH₂, —CONH-lower alkyl, —CONH-lower alkenyl, —CONH-lower alkynyl,—CONH-lower cycloalkyl, —CONH-aryl, —CONH-lower cycloalkenyl,—CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO₂-lower alkyl, —OCO₂-loweralkenyl, —OCO₂-lower alkynyl, —OCO₂-lower cycloalkyl, —OCO₂-aryl,—OCO₂-lower cycloalkenyl, —OCO₂-heteroaryl, —OCO₂-heterocycloalkyl,—OCONH₂, —OCONH-lower alkyl, —OCONH-lower alkenyl, —OCONH-lower alkynyl,—OCONH-lower cycloalkyl, —OCONH-aryl, —OCONH-lower cycloalkenyl,—OCONH-heteroaryl, —OCONH-heterocycloalkyl, —NHC(O)-lower alkyl,—NHC(O)-lower alkenyl, —NHC(O)-lower alkynyl, —NHC(O)-lower cycloalkyl,—NHC(O)-aryl, —NHC(O)-lower cycloalkenyl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂-lower alkyl, —NHCO₂-lower alkenyl,—NHCO₂-lower alkynyl, —NHCO₂-lower cycloalkyl, —NHCO₂-aryl, —NHCO₂-lowercycloalkenyl, —NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH-lower alkyl, —NHC(O)NH-lower alkenyl, —NHC(O)NH-lower alkynyl,—NHC(O)NH-lower cycloalkyl, —NHC(O)NH-aryl, —NHC(O)NH-lowercycloalkenyl, —NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl,—NHC(S)NH₂, —NHC(S)NH-lower alkyl, —NHC(S)NH-lower alkenyl,—NHC(S)NH-lower alkynyl, —NHC(S)NH-lower cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-lower cycloalkenyl, —NHC(S)NH-heteroaryl,—NHC(S)NH-heterocycloalkyl, —S(O)-lower alkyl, —S(O)-lower alkenyl,—S(O)-lower alkynyl, —S(O)-lower cycloalkyl, —S(O)-aryl, —S(O)-lowercycloalkenyl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl, —CH₂SO₂CH₃,—SO₂NH₂, —SO₂NH-lower alkyl, —SO₂NH-lower alkenyl, —SO₂NH-lower alkynyl,—SO₂NH-lower cycloalkyl, —SO₂NH-aryl, —SO₂NH-lower cycloalkenyl,—SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl, —NHSO₂-lower alkyl,—NHSO₂-lower alkenyl, —NHSO₂-lower alkynyl, —NHSO₂-lower cycloalkyl,—NHSO₂-aryl, —NHSO₂-lower cycloalkenyl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —S-lower alkenyl, —S-lower alkynyl, —S-lowercycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl, ormethylthiomethyl.

The term “heterocycloalkyl” as used herein specifically refers toazetidinyl, [1,3]dioxolane, pyrazolinyl, pyrrolidinyl, 2-pyrrolinyl,3-pyrrolinyl, tetrahydrofuranyl, dihydrofuranyl, pyranyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothienyl, imidazolinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, isothiazolidinyl, piperidinyl, homopiperidinyl,quinuclidinyl, piperazinyl, lower alkyl piperazinyl, morpholinyl,thiamorpholinyl, 1-pyrazolidinyl, azepinyl; and heterocycloalkyls asdefined herein with substitutions by independent replacement of ahydrogen atom on a ring nitrogen specifically selected from the groupconsisting of lower alkyl, lower alkenyl, and lower haloalkyl; andspecifically substitutions to these heterocycloalkyls by independentreplacement of one or two of the hydrogen atoms thereon withsubstituents specifically selected from the group consisting of halide,lower haloalkyl, amino, hydroxyl, lower alkoxy, lower aminoalkyl, lowerhydroxyalkyl, nitrile, lower alkyl nitrile, lower alkyl, lower alkenyl,lower alkynyl, carboxyl, amide, hydroxyamide, lower alkoxyalkyl, loweralkoxycarbonyl, lower alkylcarbonylamino, —SH, and thioalkyl. Forexample, substitutions to pyrrolidinyl may includehydroxyl-pyrrolidinyl, chloropyrrolidinyl, methoxy-pyrrolidinyl,nitrile-pyrrolidinyl, methyl-pyrrolidinyl, and amino-pyrrolidinyl.

The term “heteroatom” as used herein refers to a nitrogen, sulfur, oroxygen atom.

The term “lower thioalkyl” as used herein by itself or as part ofanother substituent, means a radical of the formula —SR, wherein R is alower alkyl or lower cycloalkyl group as defined herein. Examples oflower alkylthio groups include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, butylthio, and tert-butylthio.

The term “carboxy,” as used herein, means a group of formula —COOH.

The term “hydroxy,” as used herein, means a group of formula —OH.

The phrase “hydroxy protecting group,” as used herein, means a labilechemical moiety which is known in the art to protect a hydroxyl groupagainst undesired reactions during synthetic procedures. Following suchprocedures, the hydroxy protecting group may be selectively removed.Examples of hydroxy protecting groups include, but are not limited to,methylthiomethyl, tert-butyldimethylsilyl, tertbutyldiphenylsilyl, acylsubstituted with an aromatic group, and the like.

The phrase “protected hydroxy,” as used herein, means a hydroxy groupprotected with a hydroxy protecting group, as defined above, includingbenzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups,for example.

The phrase “amino protecting group,” as used herein, means a labilechemical moiety which is known in the art to protect an amino groupagainst undesired reactions during synthetic procedures. Following suchprocedures, the amino protecting group may be selectively removed.Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

The phrase “protected amino,” as used herein, means an amino groupprotected with an amino protecting group as defined above.

In accordance with the present invention, compounds as disclosed hereinare surprisingly effective at inhibiting or reducing the formation orgrowth of biofilms. Furthermore, it is also disclosed that theco-administration to a bacterial biofilm of compounds described hereinwith an antimicrobial agent, antibiotic, or biocide provides increasedsusceptibility of the bacteria within the biofilm to the antimicrobialagent, antibiotic, or biocide. The instant invention thus provides fornovel compounds, compositions, compositions comprising biofilminhibitors and antimicrobial agents or antibiotics or biocides, andvarious methods of using the compositions containing the biofilminhibitors of the invention to reduce or inhibit the formation or growthof bacterial biofilms.

The compounds of the invention may be prepared by the techniquesdescribed in the examples below, starting from the ursane or oleananetriterpene scaffolds like ursolic acid and oleanolic acid. While atypical starting chemical compound in the semi-synthetic preparation ofthe compounds of the invention may be ursolic acid, oleanolic acid,corosolic acid, asiatic acid, or madecassic acid, oleanolic acid andursolic acid are preferred staring compounds. In designingsemi-synthetic strategies to prepare analogs of the starting chemicalcompound, modifications at certain positions of the scaffold of thebasic chemical compound prove to be important for modulating biofilminhibition, while other modifications at positions can improve thebioavailability of the compound. Many of these modifications oroptimizations are taught in the literature known to those skilled in theart, including The Organic Chemistry of Drug Design and Drug Action,2^(nd) Edition, by Richard B. Silverman, incorporated herein in itsentirety by this reference. Improvement of the bioavailability of thecompound expands the therapeutic range of the compounds by reducingcertain cellular toxicities in the subject.

Compounds of this invention include:

Com- pound Chemical Structure 1

2

3

4

5

6

7

8

9

10

11

12

62

64

65

68

84

88

90

91

92

The invention especially teaches the remarkable discovery asdemonstrated by the examples that the following Structure II

may contain a diverse variety of moieties and structures at R¹ andretain the ability to inhibit or reduce the formation or growth ofbiofilms. The examples demonstrate that a diverse group of aryl,substituted aryl, heteroaryl, and substituted heteroaryl moieties andstructures may be added at R¹ and retain potent activity to reduce orinhibit the formation or growth of biofilms. A few representativechemical structures prepared semi-synthetically and described hereinincluding the examples are as follows:

The present invention also includes compounds of the following chemicalStructure III:

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl, R³ isselected from the group consisting of hydrogen or methyl, and one of R⁴and R⁵ is hydrogen and the other is methyl. Salts are also contemplatedby the present invention as described in the examples.

Based upon the teachings described herein, those skilled in the artappreciate that Structure III substituted with any of the followingmoieties at R¹ will exhibit potent activities at inhibiting theformation of biofilms.

The present invention provides compounds of the following chemicalStructure IV

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl, whereinR³ is selected from the group consisting of hydrogen or methyl; whereinone of R⁴ and R⁵ is hydrogen and the other is methyl. Salts are alsocontemplated by the present invention as described in the examples.

Based upon the teachings described herein, those skilled in the artappreciate that Structure IV substituted with the following moieties atR¹ would exhibit potent activities at inhibiting the formation ofbiofilms:

Various pharmaceutical compositions contemplated by the presentinvention, including the compounds of the invention and the specificexamples described herein, further including pharmaceutically acceptablederivable prodrugs or prodrugs thereof. A “pharmaceutically acceptablederivable prodrug or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivable prodrug of a compoundof this invention which, upon administration to a patient, is capable ofproviding (directly or indirectly) a compound used in this invention. Itwill be recognized that the efficacy of the compounds of this inventionis related to localized reaction sites on the compounds. Accordingly, asillustrated by the examples below, a wide variety of substitutionstherefore may be made at various sites on the compounds spacially remotefrom the localized reaction sites that do not significantly interferewith the efficacy of the compounds Likewise, substitutions to formpharmaceutically acceptable salts, esters, salts of esters, and othersuch derivable prodrugs of the compounds of this invention are arecontemplated herein as well. Thus, compounds with such innocuoussubstitutions do no depart from the scope of the invention.

On the other hand, however, certain moieties have been found to be sosignificant in size or reactivity as to interfere significantly with theefficacy of the compounds. Thus, highly reactive, polar, ionic or largesubstituents such as those shown in the examples below as having adeleterious affect on the activity of the compound are are excluded fromthe most preferred embodiments of the invention.

Compounds useful in the present invention may, optionally, be convertedto their therapeutically-active non-toxic acid salt forms by treatmentwith appropriate acids. Such acids include inorganic acids, e.g.,hydrochloric and hydrobromic acids, sulfuric acid, nitric acid,phosphoric acid and like acids; or organic acids, such as acetic,propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxo-propanoic,ethanedioic, propanedioic and like acids. Of course, the salt forms maybe converted into the free base form by treatment with alkali. Thepharmaceutically-acceptable acid salts of the present invention alsocomprise the solvates that the compositions of the present invention mayform, which, of course, are included within the scope of the presentinvention. Non-limiting examples of such solvates are hydrates,alcoholates and the like.

Such pharmacologic compositions may be formulated in various ways knownin the art for administration purposes. Pharmaceutical compositions ofthe present invention can be prepared by combining an effective amountof the particular compound of this invention, typically in base or acidsalt form, as the active ingredient with one or morepharmaceutically-acceptable carriers and delivery vehicles. Numerouspharmaceutically acceptable carriers and delivery vehicles exist thatare readily accessible and well-known in the art, which may be employedto generate the preparation desired (i.e. that permit administration ofthe pharmaceutical composition orally, topically, rectally,percutaneously, by parenteral injection, intranasally or by inhalation).Representative examples of pharmaceutically acceptable carriers anddelivery vehicles include aluminum stearate, lecithin, serum proteins,such as human serum albumin; buffer substances such as the variousphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids; water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyarylates, waxes, polyethylene, polyoxypropylene-block polymers,polyethylene glycol and wool fat, and the like. Other constituents, suchas aids for taste, color, tableting, and so forth, may be combined withthe active ingredient and carrier for any of the many known purposes ofsuch additives. Examples of such additives are discussed bleow.

The pharmacologic compositions described herein may further be preparedin unitary dosage form for administration orally, percutaneously, byparenteral injection (including subcutaneous, intramuscular, intravenousand intradermal), topically, intranasally, by inhalation, or forapplication to a medical device, such as an implant, catheter, or otherdevice. In preparing the compositions that permit administration of anoral dosage, for example, any of the pharmaceutically acceptablecarriers known in the art may be used, such as water, glycols, oils,alcohols and the like in the case of carriers that permit oral deliveryof liquid preparations such as suspensions, syrups, elixirs andsolutions. When solid pharmaceutically acceptable carriers are desiredthat permit oral or rectal administration, starches, sugars, kaolin,lubricants, binders, cellulose and its derivable prodrugs, anddisintegrating agents and the like may be used to prepare, for example,powders, pills, capsules and tablets.

For pharmaceutically acceptable carriers that permit parenteraladministration, the pharmaceutically acceptable carriers often comprisesterile water, which may be supplemented with various solutes to, forexample, increase solubility. Injectable solutions may be prepared inwhich the pharmaceutically acceptable carrier comprises saline solution,glucose solution, or a mixture thereof, which may include certainwell-known anti-oxidants, buffers, bacteriostats, and other solutes thatrender the formulation isotonic with the blood of the intended patient.

For pharmaceutically acceptable carriers that permit intranasaladministration, the pharmaceutically acceptable carriers often comprisepoly acrylic acids such as Carbopol® 940, a hydrogenated castor oil suchas Cremophor® RH40, glycerol, vinylpyrrolidones such as PVP-K90® or PVPK30®, polyethylene glycols such as PEG 1450®, benzyl alcohol, Edetatesodium, hydroxycellulose, potassium chloride, potassium phosphate, andsodium phosphate. Compositions used for intranasal administration alsocommonly include benzalkonium chloride as an anti-microbialpreservative.

For pharmaceutically acceptable carriers that permit administration byinhalation, the pharmaceutically acceptable carriers often comprisesolvent/carrier/water mixtures that are easily dispersed and inhaled viaa nebulizer or inhaler. For example, a mixture of ethanol/propyleneglycol/water in the ratio of about 85:10:5 (parts ethanol: partspropylene glycol: parts water) can be used to administer the compoundsand compositions of the invention via inhalation. Ratios as expressedherein are based on parts by weight.

For pharmaceutically acceptable carriers that permit percutaneousadministration, the pharmaceutically acceptable carrier may, optionally,comprise a penetration enhancing agent and/or a wetting agent.

Dosage forms that permit topical or transdermal administration of acompound of this invention include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. The activecompound or compounds is/are mixed under sterile conditions with apharmaceutically acceptable carrier and optionally one or morepreservatives and/or buffers. In the context of certain embodiments ofthis invention, the active compound is a pentacyclic acid triterpene. Inthe context of other embodiments of this invention, the pentacyclic acidtriterpene is combined in the composition with another active compoundthat is an antimicrobial agent or antibiotic.

The ointments, pastes, creams and gels may contain, in addition to anactive compound or compounds according to the present invention,pharmaceutically acceptable carriers that permit topical or transdermaladministration such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivable prodrugs,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

In some cases, the pH of the pharmaceutical formulations contemplatedherein may be adjusted with acceptable acids, bases or buffers toenhance the stability of one or more of the active compounds present ortheir delivery forms. In the context of certain embodiments of thisinvention, the active compound is a pentacyclic acid triterpene. In thecontext of other embodiments of this invention, the pentacyclic acidtriterpene is combined in the composition with another active compoundthat is an antimicrobial agent or antibiotic.

Still further, in order to prolong the anti-bacterial effect of acompound disclosed herein, it may be desirable to slow the absorption ofthe compound from subcutaneous or intramuscular injection. This may beaccomplished using a liquid suspension of crystalline or amorphousmaterial with poor water solubility. The rate of absorption of thecompound then depends upon its rate of dissolution, which, in turn, maydepend upon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally administered drug form may be accomplishedby dissolving or suspending the compound in an oil vehicle.

Injectable depot forms are made, e.g., by forming microencapsulematrices of one or more compounds of the present invention inbiodegradable polymers such as polylactide-polyglycolide. Depending uponthe ratio of active(s) to polymer and the nature of the particularpolymer employed, the rate at which such active(s) is released may becontrolled. Examples of other biodegradable polymers includepoly(orthoesters) and poly(anhydrides). Depot injectable formulationsare also prepared by entrapping the compound in liposomes ormicroemulsions that are compatible with body tissues.

The pharmaceutical composition may also be a dentifrice. In the presentinvention, “dentifrice” is understood to broadly include compositionssuitable for administering to the oral cavity, especially, for example,to the gingival/mucosal tissue or to the teeth. Thus, the dentifrice mayinclude toothpastes, toothpowders, liquid dentifrices, mouth detergents,mouthwashes, troches, chewing gums, dental or gingival massage creams,dental strips, dental gels, and gargle tablets.

When the pharmaceutical composition of this invention is a dentifricesuch as tooth paste, a tooth or gum adherence promoting substanceselected from the group consisting of copolymers of methyl vinyl etherand maleic anhydride, copolymers of vinyl pyrrolidone and vinyl acetate,and cyclodextrins may also be included in the composition. Copolymers ofmethyl vinyl ether and maleic anhydride useful in this invention mayhave molecular weights ranging from 200,000 to 2,000,000 kD and may befree acids, mixed sodium and calcium salts, or half ester derivableprodrugs. Representative commercial sources of the copolymers of methylvinyl ether and maleic anhydride include GANTREZ® AN(CAS #9011-16-9)GANTREZ® S (CAS #25153-40-69) GANTREZ® MS (CAS#62386-95-2) GANTREZ® ES(CAS#25087-06-3 or CAS#25119-68-0) and can be obtained fromInternational Specialty Products Wayne, N.J. Copolymers of vinylpyrrolidone and vinyl acetate useful in the invention typically have amolecule weight of approximately 27,000 kD and are water soluble.Representative commercial sources of the copolymers of vinyl pyrrolidoneand vinyl acetate PLASDONE® S-630 and can be obtained from InternationalSpecialty Products Wayne, N.J. Cyclodextrins useful in the invention arecyclic oligosaccharides composed of either 6, 7 or 8 glucose units (a-,b- and g-cyclodextrin, respectively). Representative commercial sourcesof the cyclodextrins useful in this invention include CAVAMAX® W6Pharma, CAVAMAX® W7 Pharma and CAV AMAXW8 Pharma (a-, b- andg-cyclodextrin, respectively) and can be obtained from InternationalSpecialty Products Wayne, N.J.

When the composition of this invention is a dentifrice, an antimicrobialagent is selected from the group consisting of triclosan, metronidazole,tetracyclines, quinolones, plant essential oils, camphor, thymol,carvacrol, menthol, eucalyptol, and methyl salicylate may also beincluded. Pharmaceutically acceptable carriers that permitadministration of the pentacyclic acid triterpene compounds of thisapplication as dentifrices include sorbitol, glycerin, silica, sodiumlauryl sulfate and Xanthum gum. The dentifrices of this invention mayalso include sodium fluoride.

Reducing or Inhibiting Bacterial Biofilms in Plants

Finally, bacterial biofilms also may be reduced or inhibited by theapplication or administration of compositions containing compoundsdisclosed herein when the subject is a plant. Thus, the compound or acomposition containing an active compound described herein may beadministered to a plant, such as a surface of a plant to reduce orinhibit the formation of a biofilm on the plant.

It is believed that the methods described herein are applicable toreducing or inhibiting a variety of bacterial biofilms in plants.Pseudomonadaceae, Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceaeand Streptomycetaceae bacteria are all economically significant plantpathogens that may be controlled by the present invention. Non-limitingexamples of specific plant pathogens involving biofilms that may beeffectively inhibited by the methods described herein include:Xanthomonas species, such as, for example, Xanthomonas campestris pv.oryzae; Pseudomonas species, such as, for example, Pseudomonas syringaepv. lachrymans; and Erwinia species, such as, for example, Erwiniaamylovora. It is also believed that the compositions used in the methodsof reducing or inhibiting bacterial biofilms of plants described hereincan further comprise antimicrobial agents such as bronopol,dichlorophen, nitrapyrin, nickel dimethyldithiocarbamate, kasugamycin,octhilinone, furancarboxyl, oxytetracyclin, probenazole, streptomycin,tecloftalam, copper sulphate and other copper preparations.

Methods of reducing or inhibiting bacterial biofilms described hereincan be used to treat all plants and parts of plants. By reference to“plants,” what is meant here is all plants and plant populations such asdesired and undesired wild plants or crop plants (including naturallyoccurring crop plants). Crop plants can be plants obtainable byconventional breeding and optimization methods or by biotechnologicaland genetic engineering methods or combinations of these methods, andinclude the transgenic plants and the plant varieties that can or cannotbe protected by varietal property rights. The phrase “parts of plants”as used herein is to be understood as meaning all above-ground andbelow-ground parts and organs of plants, such as shoot, leaf, flower androot, examples which may be mentioned being leaves, needles, stems,trunks, flowers, fruit-bodies, fruits and seeds and also roots, tubersand rhizomes. Parts of plants also include harvested plants andvegetative and generative propagation material, for example, seedlings,tubers, rhizomes, cuttings and seeds.

The treatment of the plants and the parts of plants with the activecompounds according to the invention is carried out directly or byaction on their surroundings, habitat or storage space, according tocustomary treatment methods, for example by dipping, spraying,evaporating, atomizing, broadcasting, spreading-on and, in the case ofpropagation material, in particular in the case of seeds, furthermore byone- or multi-layer coating.

Agriculturally Acceptable Carriers and Compositions

Depending on their particular physical and/or chemical properties, thecompounds and compositions described herein can be converted to thecustomary formulations, such as solutions, emulsions, suspensions,powders, foams, pastes, granules, aerosols and microencapsulations inpolymeric substances and in coating compositions for seeds, and ULV cooland warm fogging formulations.

The following examples illustrate various aspects of the presentinvention and are not intended to limit the scope of the presentinvention. Those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example I Specificity of the Biofilm Growth Assay

Table 1, below, shows experimental results illustrating remarkableproperties of Compound 1 to inhibit the spreading of clinical isolatesof P. aeruginosa and E. coli bioflms as determined in a biofilm growthassay. The related scaffolds and analogs shown in Table 1 have beenfound to exhibit significantly less inhibitory activities than doesCompound 1 in the biofilm growth assay even though certain of thecompounds consist of chemical structures similar to that of Compound 1.These data demonstrate the novel and unique aspects of Compound 1 andits discovery and the specificity of the biofilm growth assay toidentify Compound 1 as a potent inhibitor of spreading gram-negativebacterial biofilms.

The biofilm growth assay was carried out to measure swarming or biofilmspreading on semi-solid agar media in round plates (for example, 100×15mm), also referred to as Petri dishes. For E. coli, the plates containedLB medium with 0.6% agar and 0.5% glucose. P. aeruginosa plates weremade with M8 agar supplemented with 0.2% glucose, 100 μM CaCl₂, 1 mMMgSO₄, and 0.5% Casamino acids. The agar plates of this assay consistedof 0.4% to 0.7% agar (Bacto or Noble agar), although other media mayalso be used as described in the literature and known to those skilledin the art. Test compounds at the desired concentration were added tothe cooled, autoclaved media. Portions of the media were then pouredinto the plates (20 mL/plate), and the plates allowed to dry at roomtemperature for approximately 3 to 4 hours. Alternatively, however,plates may be dried in a laminar flow hood for approximately fifteen totwenty minutes. Overnight cultures were grown in a 37° C. shaker in LB(E. coli) or TSB (P. aeruginosa). Plates were inoculated by placing 5 μLof the appropriate overnight culture in the centers of the plates. Theplates then were incubated overnight at 37° C. The area of the zone ofspreading bacteria was then measured and replicates are averaged.Percent inhibition was calculated as 100×(area without compound−areawith compound)/(area without compound). An active compound wasconsidered that which reduces the area of the spreading biofilm comparedto negative controls by greater than or equal to 85%. The values shownin Table 1 are the concentrations of compounds (μg/ml) tested in thebiofilm growth assay that reduce the area of the spreading biofilmcompared to negative controls by greater than or equal to 85% unlessnoted differently in Table 1. A >(“greater than” symbol) as used inTable 1 is well understood to those skilled in the art as an indicationthat the listed concentration was not effective at this concentration.

This example demonstrates the reduction of virulence of gram-negativebacteria. Inhibition or reduction of the growth of biofilms reducesvirulence of gram-negative bacteria. Adhesion, biofilm growth, invasion,and the secretion of enzymes or toxins contribute to virulence ofgram-negative bacteria. It is well known to those skilled in the artthat biofilms increase the virulence of gram-negative bacteria and anupregulation of virulence factors (i.e. enzymes, toxins) has beendemonstrated in gram-negative bacterial biofilms. The compounds of theinvention reduce virulence of gram-negative bacteria.

TABLE 1 Related Scaffolds and Analogs of Compound 1 P. aeruginosa E.coli COMPOUND STRUCTURE (μg/ml) (μg/ml) Compound 1

0.5 1 Uvaol

>128 >8 alpha-Amyrin

>32 Not tested Lupeol

>16 >8 Betulin

16 >16 Betulinic acid

>16 >16 Oleanolic acid

4 >8 Ursolic acid

8 8 Oleanolic acid methyl ester

>16 >16 Corosolic acid

4 Not tested Maslinic acid

4 Not tested A

8 >8 B

4 8 C

0.5 0.5 D

1 4 E

2 >16 F

4 >8 G

>8 >8 H

8 >8 I

>16 8 J

>16 >16 K

>8 >8 L

>16 >16 M

8 8 N

8 8

Example II

All of the compounds shown in Tables 2, 3, 4, and 5, below, wereprepared semi-synthetically from oleanolic acid, except for Compounds 2and 3. Compound 2 was prepared from ursolic acid. Compound 3 wasprepared from hederagenin, but could also be prepared from an oleanolicacid analog with a hydroxyl at C₂₃ or C₂₄. These compounds were testedin the spreading biofilm assay according to the methods detailed inExample I, above. The values shown in Tables 2, 3, 4, and 5 are theconcentrations of compounds tested in the biofilm growth assay thatreduce the area of the spreading biofilm compared to negative controlsby greater than or equal to 85%.

TABLE 2 Compound 1 and its Analogs and their inhibitory concentrationsin the biofilm growth assay as detailed in Example I, above P.aeruginosa E. coli COMPOUND Chemical Structure (μg/ml) (μg/ml) 1

0.5 1 2

0.5 1 3

0.5 1 4

0.25 1 5

0.5 1 6

0.125 0.25 7

0.25 1 8

0.25 1 9

0.5 1 10

0.5 1 11

0.5 0.5 12

0.5 1

TABLE 3 Analogs of Compound 1

COM- P. aeruginosa E. coli POUND R₁ (μg/ml) (μg/ml) 13 —CH═CH₂ 0.5 1 14—CH₂CH₃ 0.5 2 15

0.25 1 16

1 2 17

0.5 >2 18

0.5 1 19

1 1 20

0.5 2 21

0.5 >2 22

1 >2 23

1 2 24 —Cl 0.5 1 25 —Br 0.5 >2 26

0.25 1 27

1 >2 28

1 2 29

1 1 30

2 1 31

0.5 1 32

0.5 1 33

2 4 34

1 1 35

>1 1 36

>1 2 37

>1 1 38

1 2 39

1 2 40

0.5 2 41

1 2 42

0.25 1 43

1 1 44

0.5 2 45

0.5 1 46

0.5 1 47

0.5 1 48

0.5 1 49

0.5 >1 50

2 1 51

0.5 2 52

1 2 53

0.5 2 54

1 2 55

>1 2 56

1 2 57

>2 1 58

1 2 59

0.5 >1

TABLE 4 Analogs of Compound 1

COM- P. aeruginosa E. coli POUND R₁ (μg/ml) (μg/ml) 60

0.5 1 61

0.5 0.5 62

1 1 63

4 1 64

0.5 1 65

0.25 0.25 66

0.25 1 67

1 2 68

0.5 0.5 69

1 1 70

0.5 2 71

0.5 2 72

1 >2 73

1 1 74

1 >2 75

0.5 1 76

0.5 2 77

1 2 78

>1 1 79

0.25 0.5 80

1 1 81

0.5 1 82

0.5 1 83

0.25 0.5 84

0.125 0.5 85

0.5 1 86

0.5 2 87

0.5 1 88

0.25 0.5 89

1 1 90

0.5 1

TABLE 5 Analogs of Compound 1. Compounds 91 and 92 demonstrate thatcombined modifications at the R¹ and R² positions can be prepared andretain potent inhibitory activities against biofilms. P. aeruginosa E.coli COMPOUND CHEMICAL STRUCTURE (μg/ml) (μg/ml) 91

1 1 92

0.5 2

Example III Semi-Synthesis Procedures for the Compounds Shown in ExampleII

Preparative HPLC:

Preparative HPLC was conducted using a SunFire Prep C18 OBD Column, 5μm, 19×100 mm eluting with a gradient from 90:10 (water:acetonitrile,both with 0.05% trifluoroacetic acid) to 10:90 (water:acetonitrile, bothwith 0.05% trifluoroacetic acid) over 14 minutes followed by a 2 minutehold at 10:90 (water:acetonitrile, both with 0.05% trifluoroacetic acid)with a flow rate at 25 mL/min.

CMA:

CMA=80:18:2 Methylene Chloride/Methanol/concentrated Ammonium Hydroxide

Preparation of Common Intermediate I

(i) Preparation of Ib:(4aS,6aS,6bR,12aR)-2,2,6a,6b,9,9,12a-Heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylicacid

To a mixture of oleanolic acid (Ia, 5.0 g, 10.9 mmol) and CH₂Cl₂ (200mL) was added the Dess-Martin reagent (6.0 g, 14.2 mmol) under nitrogenat room temperature. After stirring at room temperature for 1 hour, thestarting material was consumed, as indicated by TLC (1:1 hexanes:diethylether). The reaction mixture was quenched with the addition of asolution of sodium thiosulfate and NaHCO₃ (50 g sodium thiosulfate in200 mL saturated NaHCO₃ solution). The mixture was stirred at roomtemperature for 10 minutes. The layers were separated and the aqueouslayer was extracted with EtOAc (3×250 mL).

The combined organics were dried (Na₂SO₄), filtered and concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 3:1 hexanes/diethyl ether) to provide thesub-title compound (4.9 g, 99%) as a white foam.

¹H NMR (300 MHz, CDCl₃) δ 0.81 (s, 3H), 0.90-2.05 (m, 39H), 2.42-2.44(m, 1H), 2.59-2.61 (m, 1H), 2.83-2.85 (m, 1H), 5.29-5.31 (m, 1H). ESI MSm/z 455 [C₃₀H₄₆O₃+H]⁺.

(ii) Preparation of Ic: (4aS,6aS,6bR,12aR)-Benzyl2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of Ib (4.9 g, 10.8 mmol), benzyl bromide (1.9 mL, 16.2 mmol),K₂CO₃ (2.2 g, 16.2 mmol) and DMF (185 mL) was stirred at roomtemperature for 2.5 hours. The solvent then was removed under reducedpressure and the residue was partitioned between H₂O (250 mL) and EtOAc(250 mL). The layers were separated and the aqueous layer was extractedwith EtOAc (200 mL). The combined organics were dried (Na₂SO₄) andfiltered. The solvent was removed under reduced pressure and the residuewas purified by column chromatography (silica, 3:1 hexanes/diethylether) to provide the sub-title compound (4.7 g, 81%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.65 (s, 3H), 0.89 (s, 3H), 0.92 (s, 3H),1.09-2.05 (m, 32H), 2.36-2.38 (m, 1H), 2.49-2.51 (m, 1H), 2.87-2.89 (m,1H), 5.07-5.09 (m, 2H), 5.29-5.31 (m, 1H), 7.29-7.38 (m, 5H). ESI MS m/z545 [C₃₇H₅₂O₃+H]⁺.

(iii) Preparation of Id: (4aS,6aS,6bR,12aR)-Benzyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A solution of diisopropylamine (31.8 mL, 227 mmol) and THF (250 mL) wascooled to −78° C. under nitrogen. A solution of n-butyllithium (2.5 M inhexanes, 100 mL, 251 mmol) was slowly added, maintaining the internaltemperature below −70° C. The solution was allowed to stir for 30 minand was then slowly added to a solution of Ic (65.0 g, 120 mmol) and THF(1.4 L) at −78° C. under nitrogen. This solution was stirred for 30 minafter which time a suspension of p-toluenesulfonyl cyanide (43.3 g, 239mmol) and THF (200 mL) was added over 45 min. The solution was stirredfor 10 min and then quenched by the addition of saturated ammoniumchloride solution (250 mL) at −78° C. The mixture was allowed to warm toroom temperature overnight. The organic layer was separated and theaqueous layer was extracted with EtOAc. The combined organic layers weredried (Na₂SO₄) and filtered. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,1:1 hexanes/EtOAc) to provide the sub-title compound (58 g, 85%) as awhite solid.

¹H NMR (300 MHz, CDCl₃) δ 0.65 (s, 3H), 2.14-2.16 (m, 39H), 2.93-2.95(m, 1H), 5.06-5.08 (m, 2H), 5.29-5.31 (m, 1H), 7.29-7.37 (m, 5H).

(iv) Preparation of I: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A solution of Id (2.0 g, 3.5 mmol) and hydrazine (0.33 mL, 10.6 mmol) inEtOH (18 mL) was heated at reflux for 16 hours. The solvent and excesshydrazine were removed under reduced pressure. The residue was purifiedby column chromatography (silica, 0-7% MeOH in CH₂Cl₂) to provide thetitle compound (1.8 g, 88%) as an off-white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.69 (s, 3H), 0.85-2.03 (m, 37H), 2.24-2.35(m, 1H), 2.92-2.98 (m, 1H), 5.06-5.08 (m, 2H), 5.22-5.24 (m, 1H),7.28-7.37 (m, 5H). APCI MS m/z 584 [C₃₈H₅₃N₃O₂+H]⁺.

Preparation of Common Intermediate II

(i) Preparation of IIb: (4aS,6aS,6bR,10S,12aR)-Methyl10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

Oleanolic acid (20 g, 43.79 mmol) was dissolved in DMF (200 mL).Potassium carbonate (9.06 g, 65.68 mmol) and methyl iodide (3.3 mL,52.55 mmol) were added. The mixture was stirred at room temperatureovernight and then poured into H₂O (500 mL). The precipitate wascollected by filtration. The precipitate was dissolved in EtOAc (500mL). The solution was washed with brine then dried (Na₂SO₄), filteredand concentrated to give the sub-title compound (20.9 g, 100%) which wasused without further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.70-1.97 (m, 42H), 2.83-2.84 (m, 1H),3.19-3.22 (m, 1H), 3.62 (s, 3H), 5.28 (t, J=3.6 Hz, 1H).

(ii) Preparation of IIc: (4aS,6aS,6bR,10S,12aR)-Methyl10-acetoxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of IIb (20.9 g, 44.47 mmol) in pyridine (120 mL) was addedDMAP (100 mg, 0.81 mmol) and acetic anhydride (5.04 mL, 53.36 mmol). Themixture was stirred at room temperature for 20 hours and poured into H₂O(300 mL). The precipitate was collected by filtration. The precipitatewas dried in a vacuum oven overnight to obtain the sub-title compound(21.33 g, 94%) which was used without further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.72-1.97 (m, 43H), 2.04 (s, 3H), 2.82-2.88(m, 1H), 3.62 (s, 3H), 4.46-4.51 (m, 1H), 5.27 (t, J=3.6 Hz, 1H).

(iii) Preparation of IId: (4aR,6aS,6bR,10S,12aS)-Methyl10-acetoxy-13,14,14b-tribromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of IIc (35 g, 68.35 mmol) in AcOH (330 mL) and CCl₄ (160mL) was added bromine (14.05 mL, 273.4 mmol) slowly. The mixture wasstirred at room temperature for 17 hours. The CCl₄ was removed underreduced pressure and the reaction mixture was poured into H₂O (1 L). Theprecipitate was collected by filtration and then dissolved in EtOAc (1L). The solution was washed with brine then dried (Na₂SO₄), filtered andconcentrated to give the sub-title compound (50.5 g, 98%) which was usedwithout further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.75-1.75 (m, 38H), 2.04 (s, 3H), 2.40-2.43(m, 2H), 2.57 (t, J=12.6 Hz, 1H), 3.63 (s, 3H), 4.44-4.50 (m, 1H),4.58-4.64 (m, 1H).

(iv) Preparation of IIe: (4aS,6aS,6bR,10S,12aR)-Methyl10-acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

Zinc dust (21.8 g, 335.7 mmol) was added to a solution of IId (50.5 g,67.15 mmol) and AcOH (450 mL). The mixture was stirred at roomtemperature overnight and then heated at reflux for 2 hours. Thereaction mixture was cooled to room temperature and then poured into H₂O(600 mL). The precipitate was collected by filtration and then dissolvedin EtOAc (1 L). The solution was washed with brine then dried (Na₂SO₄),filtered and concentrated. The residue was purified by columnchromatography (silica, 0-10% EtOAc in hexanes) to obtain the sub-titlecompound (21.5 g, 54%).

¹H NMR (300 MHz, CDCl₃) δ 0.75-1.78 (m, 40H), 1.93-1.95 (m, 1H), 2.04(s, 3H), 2.41 (m, 2H), 3.54 (m, 1H), 3.63 (s, 3H), 4.48 (m, 1H).

(v) Preparation of IIf:(4aS,6aS,6bR,10S,12aR)-10-Acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylicacid

To a solution of IIe (21.5 g, 36.31 mmol) in 2,6-lutidine (300 mL) wasadded lithium iodide (72.9 g, 544.7 mmol). The mixture was heated at143° C. overnight and then cooled to room temperature. The mixture wasacidified (pH≈4) with aqueous HCl and extracted with EtOAc (300 mL×3).The combined extracts were washed with H₂O and brine then dried(Na₂SO₄), filtered and concentrated to afford the sub-title compound(22.8 g, 109%) which was used without further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.80-1.82 (m, 42H), 2.04 (s, 3H), 2.41 (m,2H), 3.51 (m, 1H), 4.48 (m, 1H).

(vi) Preparation of IIg: (4aS,6aS,6bR,10S,12aR)-Benzyl10-acetoxy-14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of IIf (21.2 g, 36.67 mmol) and DMF (350 mL) was addedK₂CO₃ and BnBr (as used herein, “Bn” represents benzyl). The reactionmixture was stirred overnight at room temperature and then poured intoH₂O (1 L). The precipitate was collected by filtration and thendissolved in EtOAc (1 L). The solution was washed with H₂O and brinethen dried (Na₂SO₄), filtered and concentrated to afford the sub-titlecompound (24.5 g, 100%) which was used without further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.57-2.00 (m, 41H), 2.04 (s, 3H), 2.34 (d,J=8.7 Hz, 2H), 3.59 (dd, J=4.8, 4.2 Hz, 1H), 4.49 (m, 1H), 5.08 (s, 2H),7.29-7.38 (m, 5H).

(vii) Preparation of IIh: (4aS,6aS,6bR,10S,12aR)-Benzyl14-bromo-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of IIg (24.5 g, 36.67 mmol) in MeOH (500 mL) was addedpotassium hydroxide (8.2 g, 146.7 mmol) and the mixture was heated atreflux for 2.5 hours. The resulting mixture was concentrated and theresidue was dissolved in EtOAc (1 L). The solution was washed with H₂Oand brine then dried (Na₂SO₄), filtered and concentrated. The residuewas purified by column chromatography (silica, 0-30% EtOAc in hexanes)to afford the sub-title compound (21.3 g, 93%).

¹H NMR (300 MHz, CDCl₃) δ 0.58-1.81 (m, 41H), 2.01 (m, 1H), 2.37 (d,J=9.3 Hz, 2H), 3.21 (m, 1H), 3.58 (dd, J=4.8, 4.1 Hz, 1H), 5.08 (s, 2H),7.29-7.38 (m, 5H).

(viii) Preparation of IIi: (4aS,6aS,6bR,12aR)-Benzyl14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylatelate

To a solution of IIh (21.3 g, 34.02 mmol) in CH₂Cl₂ (400 mL) was addedthe Dess-Martin reagent (18.76 g, 44.23 mmol). The mixture was stirredat room temperature for 5 hours, quenched with aqueous sodiumthiosulfate and NaHCO₃ and then extracted with EtOAc (3×300 mL). Theorganic solution was washed with H₂O and brine then dried (Na₂SO₄),filtered and concentrated. The residue was purified by columnchromatography (silica, 0-15% EtOAc in hexanes) to afford the sub-titlecompound (19.3 g, 90%).

¹H NMR (300 MHz, CDCl₃) δ 0.24-2.27 (m, 40H), 2.94 (d, J=8.7 Hz, 1H),3.44 (dd, J=4.8, 4.2 Hz, 1H), 5.13-5.15 (m, 2H), 7.33-7.35 (m, 5H).

(ix) Preparation of IIj: (4aS,6aS,6bR,12aR)-Benzyl14-bromo-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.64 mL, 4.5 mmol) in THF (15 mL) wasadded n-butyl lithium (2.5 M; 1.9 mL, 4.8 mmol) at −78° C. The mixturewas stirred at −78° C. for 10 min. The LDA solution was added to asolution of IIi (1.5 g, 2.4 mmol) in THF (40 mL) pre-cooled to −78° C.The mixture was stirred at −78° C. for 1 hour. A solution of4-methylbenzenesulfonyl cyanide (815 mg, 4.5 mmol) in THF (5 mL) wasadded and stirred for 30 min at −78° C. The reaction mixture was warmedto −20° C. over 1 hour. The reaction mixture was quenched with saturatedNH₄Cl solution and extracted with EtOAc (3×50 mL). The organic layer waswashed with H₂O and brine then dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (silica,0-20% EtOAc in hexanes) to afford the sub-title compound (1.2 g, 77%).

¹H NMR (300 MHz, CDCl₃) δ 0.56-2.42 (m, 41H), 3.61-3.89 (m, 2H), 5.08(s, 2H), 7.35 (m, 5H).

(x) Preparation of II: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-bromo-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of IIj (6.4 g, 9.89 mmol) and EtOH (30 mL) was addedhydrazine (0.62 mL, 19.78 mmol). The solution was heated at refluxovernight. The reaction mixture was concentrated and then the residuewas purified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the title compound (5.0 g, 76%).

¹H NMR (300 MHz, CDCl₃) δ 0.63-2.06 (m, 38H), 2.29 (d, J=14.7 Hz, 1H),2.45 (d, J=9.0 Hz, 2H), 3.65 (dd, J=4.4, 4.2 Hz, 1H), 5.08 (s, 2H),7.29-7.39 (m, 5H). APCI MS (Positive Mode) m/z 662 [C₃₈H₅₂BrN₃O₂+H]⁺.

Preparation of Common Intermediate III

(i) Preparation of III:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonylchloride

To a suspension of 1 (as defined above) (3.0 g, 6.08 mmol) in CH₂Cl₂ wasadded thionyl chloride (4.4 mL, 60.85 mmol) at room temperature. Themixture was stirred for 2.5 hours, after which time the solvent wasremoved under reduced pressure. The residue was dried in a high vacuumto provide the title compound (3.2 g, >100%). The material was usedwithout further purification. APCI MS (Positive Mode) m/z 512[C₃₁H₄₆ClN₃O+H]⁺.

(i) Preparation of 1:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A suspension of I (as defined above) (1.8 g, 3.1 mmol), 10% Pd/C (1.0 g)and MeOH (100 mL) was stirred under hydrogen at atmospheric pressure for2 hours. The catalyst was removed by filtration through diatomaceousearth and the filtrate was concentrated under reduced pressure toprovide the title compound (1.4 g, 88%) as an off-white solid.

R_(f) 0.37 (9:1 Methylene Chloride/Methanol) m.p. 263-266° C.

¹H NMR (500 MHz, CD₃OD) δ 0.82-0.98 (m, 9H), 1.12-1.31 (m, 15H),1.39-1.87 (m, 13H), 2.01-2.10 (m, 3H), 2.35-2.42 (m, 1H), 2.87-2.91 (m,1H), 5.28-5.33 (m, 1H). ESI MS m/z 494 [C₃₁H₄₇N₃O₂+H]⁺. HPLC 98.8% (area%), t_(R)=16.2 min.

Example 2

(i) Preparation of 2b:(4aS,6aS,6bR,12aR)-1,2,6a,6b,9,9,12a-Heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylicacid

To a mixture of ursolic acid (2a, 1.0 g, 2.18 mmol) and CH₂Cl₂ (55 mL)was added the Dess-Martin reagent (1.2 g, 2.83 mmol) under nitrogen atroom temperature. After stirring at room temperature for 2.5 hours, thestarting material was consumed as indicated by TLC (1:1 hexanes:diethylether). The reaction mixture was quenched with the addition of asolution of sodium thiosulfate and NaHCO₃ (6.3 g sodium thiosulfate in25 mL saturated NaHCO₃ solution). The mixture was stirred at roomtemperature for 10 minutes. The layers were separated and the aqueouslayer was extracted with EtOAc (3×250 mL). The combined organics weredried (Na₂SO₄), filtered and concentrated under reduced pressure. Theresidue was purified by column chromatography (silica, 3:1hexanes/diethyl ether) to provide the sub-title compound (924 mg, 92%)as a white foam solid.

(ii) Preparation of 2c: (4aS,6aS,6bR,12aR)-Benzyl1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 2b (924 mg, 2.03 mmol), benzyl bromide (0.31 mL, 0.64mmol), K₂CO₃ (393 mg, 2.84 mmol) and DMF (50 mL) was stirred at roomtemperature for 12 hours. The solvent was then removed under reducedpressure and the residue was partitioned between H₂O (150 mL) and EtOAc(150 mL). The layers were separated and the aqueous layer was extractedwith EtOAc (50 mL). The combined organics were dried (Na₂SO₄) andfiltered. The solvent was removed under reduced pressure and the residuewas purified by column chromatography (silica, 1:1 hexanes/diethylether) to provide the sub-title compound (1.0 g, 92%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.68 (s, 3H), 0.84 (d, J=6.3 Hz, 3H), 0.92 (d,J=6.0 Hz, 3H), 1.02 (s, 3H), 1.03 (s, 3H), 1.08 (s, 6H), 1.20-2.05 (m,20H), 2.26 (d, J=15.0 Hz, 1H), 2.30 (m, 1H), 2.50 (m, 1H), 4.96 (m, 2H),5.24 (m, 1H), 7.30-7.35 (m, 5H).

(iii) Preparation of 2d: (4aS,6aS,6bR,12aR)-Benzyl11-cyano-1,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A solution of diisopropylamine (0.45 mL, 3.24 mmol) and THF (5 mL) wascooled to −78° C. under nitrogen. A solution of n-butyllithium (2.5 M inhexanes, 1.43 mL, 3.56 mmol) was slowly added, maintaining the internaltemperature below −70° C. The solution was allowed to stir for 30 minand was then slowly added to a solution of 2c (880 mg, 1.62 mmol) andTHF (10 mL) at −78° C. under nitrogen. This solution was stirred for 30min after which time a suspension of p-toluenesulfonyl cyanide (587 mg,3.24 mmol) and THF (2 mL) was added over 45 min. The solution wasstirred for 10 min and then quenched by the addition of saturatedammonium chloride solution (3 mL) at −78° C. The mixture was allowed towarm to room temperature overnight. The organic layer was separated andthe aqueous layer was extracted with EtOAc. The combined organic layerswere dried (Na₂SO₄) and filtered. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,3:1 hexanes/Et₂O) to provide the sub-title compound (600 mg, 65%) as awhite foam solid.

(iv) Preparation of 2e: (4aS,6aS,6bR,13aR)-Benzyl12-amino-1,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A solution of 2d (303 mg, 0.53 mmol) and hydrazine (0.050 mL, 1.59 mmol)in EtOH (5 mL) was heated at reflux for 12 hours. The solvent and excesshydrazine were removed under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to provide thesub-title compound (234 mg, 75%) as a white foam solid.

¹H NMR (300 MHz, CDCl₃) δ 0.69 (s, 3H), 0.86 (d, J=5.7 Hz, 3H), 0.88 (s,3H), 0.93 (d, J=6.0 Hz, 3H), 1.09 (s, 3H), 1.13 (s, 3H), 1.22 (s, 3H),1.38-2.05 (m, 19H), 2.28 (m, 1H), 2.30 (d, J=14.7 Hz, 1H), 4.96 (q,J=12.6 Hz, 2H), 5.29 (s, 1H), 7.32 (m, 5H).

(v) Preparation of 2:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A suspension of 2e (234 mg, 0.40 mmol), 10% Pd/C (60 mg) and MeOH (6 mL)was stirred under hydrogen at atmospheric pressure for 9 hours. Thecatalyst was removed by filtration and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-15% MeOH in CH₂Cl₂) to provide the titlecompound (103 mg, 52%) as an off-white solid.

R_(f) 0.16 (89:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

m.p. 258-278° C. ¹H NMR (300 MHz, CD₃OD) δ 0.89 (s, 9H), 0.91 (s, 3H),1.15 (s, 6H), 1.19 (s, 3H), 2.05-2.10 (m, 19H), 2.22 (m, 1H), 2.40 (m,1H), 5.30 (s, 1H). ESI MS m/z 494 [C₃₁H₄₇N₃O₂+H]⁺.

Example 3

(i) Preparation of 3b: (4aS,6aS,6bR,8aR,9R,10S,12aR,14bS)-Benzyl10-hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 3a (2.0 g, 4.2 mmol), benzyl bromide (0.6 mL, 5.0 mmol) andK₂CO₃ (580 mg, 6.3 mmol) in DMF (20 mL) was stirred at room temperaturefor 12 hours. The reaction mixture was diluted with EtOAc (300 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-50% EtOAc in hexanes) to afford the sub-titlecompound (2.3 g, 95%).

(ii) Preparation of 3c: (4aS,6aS,6bR,8aR,9R,12aR,14bS)-Benzyl9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of 3b (400 mg, 0.71 mmol) in benzene (20 mL) was addedRuCl₂(PPh₃)₃ (682 mg, 0.71 mmol). The mixture was stirred at roomtemperature for 12 hours. The reaction was not complete. An additionalamount of RuCl₂(PPh₃)₃ (341 mg, 0.36 mmol) was added and the reactionmixture was continued to stir for 8 hours. The reaction mixture wasdiluted with EtOAc (150 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-50% EtOAc in hexanes)to afford the sub-title compound (150 mg, 38%).

(iii) Preparation of 3d: (4aS,6aS,6bR,8aS,9S,12aS,14bS)-Benzyl2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 3c (150 mg, 0.26 mmol) and K₂CO₃ (44 mg, 0.32 mmol) in MeOH(10 mL) was stirred at room temperature for 24 hours. The reactionmixture was diluted with EtOAc (150 mL) and washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-30% EtOAc in hexanes) to afford thesub-title compound (70 mg, 49%).

(iv) Preparation of 3e: (4aS,6aS,6bR,8aS,9S,12aS,14bS)-Benzyl11-cyano-2,2,6a,6b,9,12a-hexamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.073 mL, 0.52 mmol) in THF (4 mL)was added n-butyllithium (0.22 mL, 2.5 M in hexanes, 0.54 mmol) at −78°C. The solution was stirred for 30 min. The LDA solution was added to 3d(150 mg, 0.27 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (98 mg, 0.54 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-30% EtOAc in hexanes) to afford the sub-titlecompound (90 mg, 58%).

¹H NMR (300 MHz, CDCl₃) δ 0.65 (s, 3H), 0.86 (s, 3H), 0.88 (s, 3H),1.0-2.0 (m, 28H), 2.35 (m, 2H), 2.92 (m, 1H), 3.61 (m, 1H), 5.15 (s,2H), 5.30 (m, 1H), 7.30 (s, 5H).

(v) Preparation of 3f: (4aS,6aS,6bR,8aS,9S,13aS,15bS)-Benzyl12-amino-2,2,6a,6b,9,13a-hexamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 3e (90 mg, 0.15 mmol) and hydrazine (0.025 mL) in EtOH (3mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (60 mg, 65%).

(v) Preparation of 3:(4aS,6aS,6bR,8aS,9S,13aS,15bS)-12-Amino-2,2,6a,6b,9,13a-hexamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 3f (60 mg, 0.10 mmol) and 10% Pd(OH)₂/C (60 mg) in MeOH (5mL) and EtOAc (5 mL) was stirred under hydrogen balloon for 5 hours. Thereaction mixture was filtered through a pad of diatomaceous earth andwashed with CMA (25 mL). The filtrate was concentrated to dryness underreduced pressure. The residue was purified by column chromatography(silica, 0-60% CMA in CH₂Cl₂) to afford the title compound (32 mg, 68%)as a brown solid.

R_(f) 0.40 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, DMSO-d₆) δ 0.78 (s, 3H), 0.80 (s, 3H), 0.91 (s, 6H),1.05 (s, 3H), 1.10-2.0 (m, 25H), 2.15 (m, 1H), 2.25 (d, J=15.0 Hz, 1H),2.80 (m, 1H), 5.24 (s, 1H), 11.4 (bs, 1H). mp>300° C. ESI MS (PositiveMode) m/z 478 [C₃₀H₄₅N₃O₂+H]⁺.

Example 4

(i) Preparation of 4b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl2,2,6a,6b,9,9,12a,14-octamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (300 mg, 0.48 mmol), trimethylboroxine (0.3 mL, 2.15mmol), Pd(PPh₃)₄ (60 mg, 0.048 mmol) and K₂CO₃ (300 mg, 2.17 mmol) inDMF (9 mL) was heated at 100° C. for 12 hours. The reaction mixture wasdiluted with EtOAc (100 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-10% EtOAc in hexanes)to afford the sub-title compound (220 mg, 82%).

(ii) Preparation of 4c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl11-cyano-2,2,6a,6b,9,9,12a,14-octamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.15 mL, 1.07 mmol) in THF (5 mL) wasadded n-butyllithium (0.45 mL, 2.5 M in hexanes, 1.1 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 4b(315 mg, 0.56 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (203 mg, 1.1 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (215 mg, 66%).

(iii) Preparation of 4d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a,15-octamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 4c (215 mg, 0.36 mmol) and hydrazine (0.060 mL, 1.85 mmol)in EtOH (3 mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (180 mg, 84%). ESI MS (Positive Mode) m/z 598[C₃₉H₅₅N₃O₂+H]⁺.

(iv) Preparation of 4:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a,15-octamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 4d (180 mg, 0.30 mmol) and 10% Pd(OH)₂/C (100 mg) in MeOH(15 mL) and EtOAc (2 mL) was stirred under hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) followed by preparativeHPLC to provide the title compound (28 mg, 18%) as an off-white solid.

R_(f) 0.62 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.85 (s, 3H), 0.90 (s, 3H), 0.93 (s, 3H), 0.99(s, 3H), 1.20 (s, 3H), 1.23 (s, 3H), 1.38 (s, 3H), 1.40-2.05 (m, 23H),2.39 (d, J=14.7 Hz, 1H). mp>300° C. ESI MS (Positive Mode) m/z 508[C₃₂H₄₉N₃O₂+H]⁺.

Example 5

(i) Preparation of 5b: (4aS,6aS,6bR,8aR,10S,12aR,14bR)-Methyl14-bromo-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIE (36.0 g, 61.0 mmol) and KOH (13.6 g, 243 mmol) in MeOH(500 mL) was heated at reflux for 3 hours. The mixture was cooled toroom temperature and concentrated to remove MeOH. The residue wasacidified with HCl (2 M) to pH 5 and extracted with EtOAc (500 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to afford the sub-title compound (34.0 g, 94%) which wasused for the next step without further purification.

¹H NMR (300 MHz, CDCl₃) δ 0.78 (s, 3H), 0.80 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.01-2.35 (m, 33H), 3.20 (m, 1H), 3.62 (s, 3H).

(ii) Preparation of 5c: (4aS,6aS,6bR,8aR,12aR,14bR)-Methyl14-bromo-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 5b (32.0 g, 61.0 mmol) and Dess-Martin reagent (31.0 g,73.0 mmol) in CH₂Cl₂ (500 mL) was stirred at room temperature for 4hours. The mixture was quenched with saturated sodium thiosulfate (50mL) and NaHCO₃ (50 mL) and extracted with EtOAc (500 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 5-10% EtOAc in hexanes) to afford thesub-title compound (26.0 g, 81%).

¹H NMR (300 MHz, CDCl₃) δ 0.80 (s, 3H), 0.90 (s, 3H), 0.98 (s, 3H), 1.02(s, 3H), 1.05 (s, 6H), 1.06-2.55 (m, 26H), 3.62 (s, 3H).

(iii) Preparation of 5d: (4a′R,6a′R,6b′S,8a′S,12a′R,14b′R)-Methyl13′-bromo-4′,4′,6a′,6b′,11′,11′,14b′-heptamethyl-2′,4′,4a′,5′,6′,6a′,6b′,7′,8′,8a′,9′,10′,11′,12′,12a′,14′,14a′,14b′-octadecahydro-1′H-spiro[[1,3]dioxolane-2,3′-picene]-8a′-carboxylate

A flask equipped with a Dean-Stark trap was charged 5c (26.0 g, 47.4mmol), ethylene glycol (7.9 mL, 142.3 mmol) and p-toluenesulfoinc acidmonohydrate (894 mg, 4.7 mmol) in benzene (500 mL). The mixture washeated at reflux overnight. The reaction mixture was cooled to roomtemperature and diluted with EtOAc (500 mL). The organic phase waswashed with saturated NaHCO₃ (50 mL) and brine (200 mL) then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% EtOAc in hexanes) to afford thesub-title compound (18.0 g, 64%).

¹H NMR (300 MHz, CDCl₃) δ 0.74 (s, 3H), 0.83 (s, 3H), 0.92 (s, 3H), 0.94(s, 3H), 0.97 (s, 3H), 0.99 (s, 3H), 1.14 (s, 3H), 1.15-2.05 (m, 21H),2.39 (d, J=9.0 Hz, 1H), 3.57 (m, 1H), 3.63 (s, 3H), 3.93 (m, 4H).

(iv) Preparation of 5e: (4a′R,6a′R,6b′S,8a′S,12a′S,14b′R)-Methyl13′-formyl-4′,4′,6a′,6b′,11′,11′,14b′-heptamethyl-2′,4′,4a′,5′,6′,6a′,6b′,7′,8′,8a′,9′,10′,11′,12′,12a′,14′,14a′,14b′-octadecahydro-1′H-spiro[[1,3]dioxolane-2,3′-picene]-8a′-carboxylate

To a solution of 5d (3.0 g, 5.1 mmol) in THF (60 mL) was addedtert-butyllithium (10 mL, 1.5 M in heptane, 15.0 mmol) at −78° C. Themixture was stirred for 20 min. n-Methylformanilide (1.8 mL, 15.2 mmol)was added. The reaction mixture was allowed to warm to −10° C. over 1hour. The reaction mixture was quenched by saturated NH₄Cl (10 mL) andextracted with EtOAc (200 mL). The organic phase was washed with brine(200 mL) then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% EtOAc inhexanes) to afford the sub-title compound (2.0 g, 73%).

¹H NMR (300 MHz, CDCl₃) δ 0.65 (s, 3H), 0.81 (s, 3H), 0.90 (s, 6H), 0.92(s, 3H), 0.99 (s, 3H), 1.12 (s, 3H), 1.25-2.18 (m, 19H), 2.65 (s, 3H),3.85 (m, 4H), 10.3 (s, 1H).

(v) Preparation of 5f: (4a′R,6a′R,6b′S,8a′S,12a′S,14b′R)-Methyl4′,4′,6a′,6b′,11′,11′,14b′-heptamethyl-13′-(prop-1-enyl)-2′,4′,4a′,5′,6′,6a′,6b′,7′,8′,8a′,9′,10′,11′,12′,12a′,14′,14a′,14b′-octadecahydro-1′H-spiro[[1,3]dioxolane-2,3′-picene]-8a′-carboxylate

To a suspension of ethyltriphenylphosphonium iodide (1.2 g, 2.96 mmol)in benzene (20 mL) was added potassium tert-butoxide (332 mg, 2.96mmol). The mixture was stirred at room temperature for 1 hour. Asolution of 5e (400 mg, 0.74 mmol) in THF (5 mL) was added. The mixturewas stirred at room temperature for 1.5 hours. The reaction mixture wasdiluted with EtOAc (100 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-10% EtOAc in hexanes)to afford the sub-title compound (380 mg, 93%) as a mixture of cis- andtrans-isomers.

(vi) Preparation of 5g: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(prop-1-enyl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 5f (571 mg, 0.92 mmol) and p-toluenesulfonic acidmonohydrate (176 mg, 0.92 mmol) in acetone (20 mL) and H₂O (2 mL) wasstirred at room temperature for 1.5 hours. The reaction mixture wasconcentrated to remove acetone under reduced pressure and extracted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% EtOAc in hexanes) to afford thesub-title compound (500 mg, 95%).

¹H NMR (300 MHz, CDCl₃) δ 0.72 (s, 1.8H), 0.81 (s, 1.2H), 0.89 (s, 3H),0.91 (s, 3H), 1.0-2.50 (m, 38H), 3.65 (s, 3H), 5.40-5.70 (m, 1H), 6.05(d, J=12.0 Hz, 0.4H), 6.53 (d, J=15.0 Hz, 0.6H).

(vii) Preparation of 5h: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl1′-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(prop-1-enyl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.26 mL, 1.87 mmol) in THF (5 mL) wasadded n-butyllithium (0.78 mL, 2.5 M in hexanes, 1.96 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 5g(500 mg, 0.98 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (339 mg, 1.87 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (210 mg, 40%).

¹H NMR (300 MHz, CDCl₃) δ 0.65-2.01 (m, 44H), 2.42-2.80 (m, 1H), 3.60(s, 3H), 4.21 (m, 1H), 5.41-5.70 (m, 1H), 6.01 (m, 1H).

(viii) Preparation of 5i: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-((E)-prop-1-enyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 5h (200 mg, 0.37 mmol) and hydrazine (0.014 mL, 0.45 mmol)in EtOH (3 mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (130 mg, 64%).

¹H NMR (300 MHz, CDCl₃) δ 0.71 (s, 1.8H), 0.81 (s, 1.2H), 0.88 (s, 3H),0.89 (s, 6H), 0.98 (s, 1.8H), 1.02 (s, 1.2H), 1.05 (s, 3H), 1.12 (s,3H), 1.16-2.35 (m, 27H), 3.65 (s, 3H), 5.50 (m, 0.4H), 5.70 (m, 0.6H),6.05 (d, J=12.0 Hz, 0.4H), 6.55 (d, J=15.0 Hz, 0.6H).

(ix) Preparation of 5:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-((E)-prop-1-enyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 5h (90 mg, 0.16 mmol) and lithium iodide (330 mg, 2.47mmol) in 2,6-lutidine (3 mL) was heated to 144° C. for 6 hours. Thereaction mixture was cooled to room temperature and neutralized with HCl(2 M) and extracted with CH₂Cl₂/i-PrOH (3:1). The organic phase wasdried (MgSO₄), filtered and concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-40% CMA in CH₂Cl₂) followed by preparative HPLC to provide the titlecompound (20 mg, 23%) as an off-white solid.

R_(f) 0.85 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.80 (s, 3H), 0.90 (s, 6H), 1.02 (s, 3H), 1.18(s, 3H), 1.20 (s, 3H), 1.28 (s, 3H), 1.30-2.30 (m, 22H), 2.52 (d, J=15.0Hz, 1H), 3.63 (m, 1H), 5.68 (dd, J=6.0, 15.0 Hz, 1H), 6.59 (d, J=15.0Hz, 1H). mp>300° C. ESI MS (Positive Mode) m/z 534 [C₃₄H₅₁N₃O₂+H]⁺.

Example 6

wherein TIPS refers to triisopropylsilyl.

(i) Preparation of 6b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1-(triisopropylsilyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (250 mg, 0.37 mmol),1-(triisopropylsilyl)-1H-pyrrol-3-ylboronic acid (303 mg, 1.13 mmol),Pd(PPh₃)₄ (43 mg, 0.037 mmol) and K₂CO₃ (203 mg, 1.47 mmol) in benzene(3.5 mL) and EtOH (1.5 mL) was sealed and heated to 120° C. by microwavefor 1 hour. The reaction mixture was diluted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (130 mg, 43%). APCI MS (Positive Mode) m/z 805[C₅₁H₇₆N₄O₂Si+H]⁺.

(ii) Preparation of 6c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 6b (130 mg, 0.16 mmol) in THF (2 mL) was addedtetrabutylammonium fluoride (0.49 mL, 1 M in THF, 0.49 mmol). Themixture was stirred at room temperature for 2 hours. The reactionmixture was diluted with EtOAc (100 mL). The organic phase was washedwith brine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (90 mg, 87%). APCI MS (PositiveMode) m/z 649 [C₄₂H₅₆N₄O₂+H]⁺.

(iii) Preparation of 6:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 6c (90 mg, 0.13 mmol) and 10% Pd(OH)₂/C (50 mg) in MeOH (12mL) and EtOAc (3 mL) was stirred under a hydrogen balloon for 5 hours.The reaction mixture was filtered through a pad of diatomaceous earthand washed with CMA (25 mL). The filtrate was concentrated to drynessunder reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the titlecompound (18 mg, 25%) as a brown solid.

R_(f) −0.26 (9:1 Methylene Chloride/Methanol).

¹H NMR (400 MHz, CD₃OD) δ 0.50 (s, 3H), 0.80 (s, 3H), 0.86 (s, 3H), 0.95(s, 3H), 1.14 (s, 3H), 1.21 (s, 3H), 1.24 (s, 3H), 1.25-2.18 (m, 19H),2.26 (s, 1H), 2.36 (d, J=14.8 Hz, 1H), 3.62 (m, 1H), 6.08 (s, 1H), 6.65(s, 1H), 9.91 (s, 1H). mp>300° C. APCI MS (Positive Mode) m/z 559[C₃₅H₅₀N₄O₂+H]⁺.

Example 7

(i) Preparation of 7b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(furan-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (250 mg, 0.377 mmol) and potassium2-furantrifluoroborate (196 mg, 1.12 mmol) in toluene (4.5 mL) and H₂O(0.5 mL) was added K₂CO₃ (207 mg, 1.5 mmol). The mixture was spargedwith nitrogen and then Pd(PPh₃)₄ (86 mg, 0.074 mmol) was added. Thereaction mixture was heated at 120° C. for 1 hour using microwaveirradiation. The solvent was concentrated under reduced pressure. Theresidue was taken up in EtOAc (40 mL) and washed with brine (3×15 mL).The organic layer was dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (200 mg, 81%). APCI MS m/z 650[C₄₂H₅₅N₃O₃+H]⁺.

(ii) Preparation of 7:(4aS,6aS,6bR,13aR)-12-Amino-15-(furan-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 7b (200 mg, 0.308 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (200 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight.

The catalyst was removed by filtration through diatomaceous earth andthe filtrate was concentrated. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (19 mg, 11%).

R_(f) 0.20 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.78 (s, 3H), 0.86-2.50 (m, 42H), 3.74 (d,J=12.1 Hz, 1H), 6.38 (s, 2H), 7.41 (s, 1H). APCI MS m/z 560[C₃₉H₄₉N₃O₃+H]⁺. m.p. 250-270° C. dec. HPLC (Method A) 98.3% (214 nm)t_(R)=16.6 min.

Example 8

(i) Preparation of 8b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1-(phenylsulfonyl)-1H-indol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (250 mg, 0.37 mmol),1-(phenylsulfonyl)-1H-indol-3-ylboronic acid (341 mg, 1.13 mmol),Pd(PPh₃)₄ (43 mg, 0.037 mmol) and cesium carbonate (491 mg, 1.50 mmol)in toluene (4.0 mL) and H₂O (1.0 mL) was sealed and heated to 120° C. bymicrowave for 1 hour. The reaction mixture was diluted with EtOAc (100mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-5% MeOH in CH₂Cl₂) to afford the sub-titlecompound (264 mg, 83%). APCI MS (Positive Mode) m/z 839[C₅₂H₆₂N₄O₄S+H]⁺.

(ii) Preparation of 8c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(1H-indol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 8b (260 mg, 0.30 mmol) and NaOH (15 mL, 2.0 M) in MeOH (10mL) and EtOH (30 mL) was heated at reflux for 24 hours. The reactionmixture was extracted with EtOAc (100 mL). The organic phase was washedwith brine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-5% MeOH inCH₂Cl₂) to afford the sub-title compound (115 mg, 53%).

(iii) Preparation of 8:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(1H-indol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture 8c (115 mg, 0.16 mmol) and 10% Pd(OH)₂/C (60 mg) in MeOH (15mL) and EtOAc (5 mL) was stirred under a hydrogen balloon for 12 hours.The reaction mixture was filtered through a pad of diatomaceous earthand washed with CMA (25 mL). The filtrate was concentrated to drynessunder reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CMA) to afford the title compound(56 mg, 76%) as a brown solid.

R_(f) 0.75 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.14 (s, 3H), 0.65 (s, 3H), 0.88 (s, 3H), 1.09(s, 3H), 1.16 (s, 3H), 1.26 (s, 3H), 1.34 (s, 3H), 1.35-2.20 (m, 19H),2.30 (d, J=14.4 Hz, 1H), 2.40 (m, 1H), 6.96 (t, J=8.0 Hz, 1H), 7.05 (t,J=8.0 Hz, 1H), 7.07 (s, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz,1H). mp>300° C. dec. APCI MS (Positive Mode) m/z 609 [C₃₉H₅₂N₄O₂+H]⁺.

Example 9

(i) Preparation of 9b: 4-((1H-Pyrrol-2-yl)methyl)morpholine

To a solution of 1H-pyrrole-2-carbaldehyde (5.0 g, 52.5 mmol) andmorpholine (5.0 mL, 57.8 mmol) in CH₂Cl₂ (160 mL) was added sodiumtriacetoxyborohydride (12.2 g, 57.8 mmol). The mixture was stirred atroom temperature for 4 hours. The reaction mixture was diluted withEtOAc (200 mL). The organic phase was washed with aqueous NaHCO₃ andbrine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (5.3 g, 61%).

¹H NMR (400 MHz, CDCl₃) δ 2.40 (s, 4H), 3.42 (s, 2H), 3.70 (m, 4H), 6.03(s, 1H), 6.09 (s, 1H), 6.74 (s, 1H), 8.30 (bs, 1H).

(ii) Preparation of 9c:4-(1-(Triisopropylsilyl)-1H-pyrrol-2-yl)methyl)morpholine

To a solution of 9b (5.3 g, 31.9 mmol) in DMF (60 mL) was added sodiumhydride (60% in mineral oil, 1.4 g, 35.1 mmol) in an ice bath. Themixture was stirred for 5 min. Chlorotriisopropylsilane (7.4 mL, 35.1mmol) was added. The reaction mixture was stirred at room temperaturefor 12 hours. The reaction mixture was diluted with EtOAc (200 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (10.0 g, 98%).

¹H NMR (400 MHz, CDCl₃) δ 1.10 (d, J=2.5 Hz, 18H), 1.62 (m, 3H), 2.40(s, 4H), 3.42 (s, 2H), 3.70 (m, 4H), 6.20 (s, 2H), 6.80 (s, 1H).

(iii) Preparation of 9d:4-((4-Bromo-1-(triisopropylsilyl)-1H-pyrrol-2-yl)methyl)morpholine

To a solution of 9c (1.0 g, 3.1 mmol) in THF (10 mL) was addedN-bromosuccinimide (552 mg, 3.1 mmol) at −78° C. The mixture was stirredat −78° C. for 2 hours and warmed to room temperature for 1 hour. Thereaction was quenched with H₂O (5 mL) and extracted with EtOAc (100 mL).The organic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% EtOAc in hexanes) to afford the sub-titlecompound (1.0 g, 83%).

¹H NMR (400 MHz, CDCl₃) δ 1.15 (d, J=2.5 Hz, 18H), 1.58 (m, 3H), 2.40(m, 4H), 3.32 (s, 2H), 3.68 (m, 4H), 6.20 (s, 1H), 6.72 (s, 1H).

(iv) Preparation of 9e:5-(Morpholinomethyl)-1-(triisopropylsilyl)-1H-pyrrol-3-ylboronic acid

To a solution of 9d (200 mg, 0.49 mmol) in THF (5 mL) was addedn-butyllithium (2.5 M in hexanes, 0.30 mL, 0.74 mmol) at −78° C. Themixture was stirred at −78° C. for 20 min. Trimethyl borate (0.28 mL,2.45 mmol) was added. The reaction mixture was stirred at −78° C. for 1hour and quenched with MeOH (4 mL). The reaction mixture was warmed toroom temperature for 1 hour and extracted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (70 mg, 39%).

¹H NMR (400 MHz, CDCl₃) δ 1.15 (d, J=2.5 Hz, 18H), 1.58 (m, 3H), 2.40(m, 4H), 3.32 (s, 2H), 3.68 (m, 4H), 6.35 (s, 1H), 7.15 (s, 1H).

(v) Preparation of 9f: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)-1-(triisopropylsilyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (220 mg, 0.33 mmol), 9e (310 mg, 0.84 mmol), Pd(PPh₃)₄(60 mg, 0.052 mmol) and K₂CO₃ (184 mg, 1.32 mmol) in toluene (4.0 mL)and H₂O (0.5 mL) was sealed and heated to 120° C. by microwave for 1hour. The reaction mixture was diluted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (250 mg, 83%).

¹H NMR (300 MHz, CDCl₃) δ 0.42 (s, 3H), 0.73 (s, 3H), 0.78 (s, 3H), 0.85(s, 3H), 1.11-2.40 (m, 55H), 3.43 (s, 3H), 3.60 (m, 6H), 5.02 (m, 2H),6.08 (s, 1H), 6.68 (s, 1H), 7.33 (m, 5H). APCI MS (Positive Mode) m/z904 [C₅₆H₈₅N₅O₃Si+H]⁺.

(vi) Preparation of 9g: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 9f (250 mg, 0.27 mmol) in THF (5 mL) was addtetrabutylammonium fluoride (0.83 mL, 1 M in THF, 0.83 mmol). Themixture was stirred at room temperature for 2 hours. The reactionmixture was diluted with EtOAc (100 mL). The organic phase was washedwith brine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (176 mg, 87%). APCI MS(Positive Mode) m/z 748 [C₄₇H₆₅N₅O₃+H]⁺.

(vii) Preparation of 9:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 9g (176 mg, 0.23 mmol) and 10% Pd(OH)₂/C (100 mg) in MeOH(15 mL) and EtOAc (5 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) to afford the titlecompound (25 mg, 17%) as an off-white solid.

R_(f) 0.50 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.55 (s, 3H), 0.78 (s, 3H), 0.86 (s, 3H), 0.94(s, 3H), 1.12 (s, 3H), 1.21 (s, 3H), 1.24 (s, 3H), 1.25-2.18 (m, 19H),2.26 (m, 1H), 2.36 (d, J=14.8 Hz, 1H), 3.49 (m, 4H), 3.52 (m, 3H), 3.67(m, 6H), 6.03 (s, 1H), 6.63 (s, 1H). mp 270-280° C. dec. ESI MS(Positive Mode) m/z 659 [C₄₀H₅₉N₅O₃+H]⁺.

Example 10

(i) Preparation of 10b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(4-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of II (300 mg, 0.45 mmol), 4-(dimethylamino)phenylboronicacid (223 mg, 1.35 mmol), benzene (4 mL) and EtOH (1 mL) was added K₂CO₃(249 mg, 1.80 mmol).

The mixture was sparged with nitrogen and then Pd(PPh₃)₄ (104 mg, 0.08mmol) was added. The reaction mixture was heated at 120° C. for 1 hourusing microwave irradiation and then concentrated under reducedpressure. The residue was dissolved in EtOAc (20 mL) and the solutionwas washed with brine then dried (Na₂SO₄), filtered and concentrated.The residue was purified (silica, 0-5% MeOH in CH₂Cl₂) to afford thesub-title compound (254 mg, 79%).

¹H NMR (300 MHz, CDCl₃) δ 0.32-2.31 (m, 41H), 2.89 (s, 6H), 3.15 (m,1H), 5.08 (m, 2H), 6.63 (d, J=8.7 Hz, 2H), 7.03 (d, J=8.7 Hz, 2H),7.34-7.37 (m, 5H). APCI MS m/z 703 [C₄₆H₆₂N₄O₂+H]⁺.

(ii) Preparation of 10:(4aS,6aS,6bR,13aR)-12-Amino-15-(4-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 10b (248 mg, 0.35 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (150 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration and the filtrate was concentrated. Purification by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) afforded the title compound(40 mg, 19%).

R_(f) 0.32 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.31-2.37 (m, 41H), 2.88 (s, 6H), 3.18 (m,1H), 6.81 (d, J=8.7 Hz, 2H), 7.13 (d, J=8.4 Hz, 2H). APCI MS m/z 613[C₃₉H₅₆N₄O₂+H]⁺. m.p.>300° C. HPLC (Method A) 97.0% (214 nm) t_(R)=13.2min.

Example 11

(i) Preparation of 11b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of II (200 mg, 0.302 mmol), 1H-indol-5-ylboronic acid (145mg, 0.90 mmol), toluene (4.5 mL) and H₂O (0.5 mL) was added cesiumcarbonate (391 mg, 1.20 mmol). The mixture was sparged with nitrogen andthen Pd(PPh₃)₄ (69 mg, 0.06 mmol) was added. The reaction mixture washeated at 120° C. for 1 hour using microwave irradiation and thenconcentrated under reduced pressure. The residue was dissolved in EtOAc(30 mL) and the solution was washed with brine then dried (Na₂SO₄),filtered and concentrated. The residue was purified by columnchromatography (silica, 0-5% MeOH in CH₂Cl₂) to afford the titlecompound (195 mg, 92%). APCI MS m/z 699 [C₄₆H₅₈N₄O₂+H]⁺.

(ii) Preparation of 11:(4aS,6aS,6bR,13aR)-12-Amino-15-(indolin-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 11b (192 mg, 0.274 mmol), MeOH (12 mL) and EtOAc (3 mL)was flushed with nitrogen and then 10% Pd/C (228 mg) was added. Themixture was flushed with nitrogen followed by hydrogen. The mixture wasstirred under hydrogen at atmospheric pressure overnight. The catalystwas removed by filtration and the filtrate was concentrated. The residuewas purified by column chromatography (silica, 0-50% CMA in CH₂Cl₂)followed by preparative HPLC to afford the title compound (7.5 mg, 5%)as a solid.

R_(f) 0.15 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.30 (s, 3H), 0.77-2.39 (m, 43H), 2.99-3.03(m, 1H), 3.34 (t, J=7.75 Hz, 2H), 3.85 (t, J=7.75 Hz, 2H), 7.35-7.44 (m,3H). APCI MS m/z 611 [C₃₉H₅₄N₄O₂+H]⁺. m.p. 170-190° C. dec. HPLC (MethodA) 97.7% (214 nm) t_(R)=12.6 min.

Example 12

(i) Preparation of 12b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (400 mg, 0.64 mmol), pyridin-3-ylboronic acid (237 mg,1.93 mmol), Pd(PPh₃)₄ (74 mg, 0.064 mmol) and cesium carbonate (629 mg,1.93 mmol) in benzene (15 mL) and EtOH (5 mL) was heated at reflux for12 hours. The reaction mixture was diluted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-50% EtOAc in hexanes) to afford the sub-titlecompound (200 mg, 48%).

¹H NMR (300 MHz, CDCl₃) δ 0.25 (s, 3H), 0.75 (s, 3H), 0.83 (s, 3H), 1.02(s, 3H), 1.04 (s, 3H), 1.09 (s, 3H), 1.22 (s, 3H), 1.23-2.02 (m, 20H),2.30-2.50 (m, 2H), 2.90 (m, 1H), 5.05 (m, 2H), 7.14 (m, 1H), 7.37 (m,5H), 7.48 (m, 1H), 8.42 (m, 2H).

(ii) Preparation of 12c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.085 mL, 0.61 mmol) in THF (3 mL)was added n-butyllithium (0.26 mL, 2.5 M in hexanes, 0.64 mmol) at −78°C. The solution was stirred for 30 min. The LDA solution was added to12b (200 mg, 0.32 mmol) in THF (5 mL). The mixture was allowed to warmto −40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (116 mg, 0.64 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-50% EtOAc in hexanes) to afford the sub-titlecompound (80 mg, 39%).

¹H NMR (300 MHz, CDCl₃) δ 0.25 (s, 3H), 0.75 (s, 3H), 0.83 (s, 3H), 1.08(s, 3H), 1.13 (s, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.23-2.25 (m, 20H),2.90 (m, 1H), 3.82 (m, 1H), 5.04 (m, 2H), 7.14 (m, 1H), 7.37 (m, 5H),7.48 (m, 1H), 8.43 (m, 2H).

(iii) Preparation of 12d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 12c (80 mg, 0.12 mmol) and hydrazine (0.020 mL) in EtOH (2mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-50% CMA in CH₂Cl₂) to afford thesub-title compound (74 mg, 93%). ESI MS (Positive Mode) m/z 661[C₄₃H₅₆N₄O₂+H]⁺.

(iv) Preparation of 12:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 12d (74 mg, 0.11 mmol) and 10% Pd(OH)₂/C (60 mg) in MeOH(10 mL) and EtOAc (2 mL) was stirred under a hydrogen balloon for 5hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-70% CMA in CH₂Cl₂) to afford the titlecompound (25 mg, 40%) as a brown solid.

R_(f) 0.30 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.81 (s, 3H), 0.87 (s, 3H), 0.97(s, 3H), 1.03 (s, 3H), 1.08 (s, 6H), 1.25-2.10 (m, 19H), 2.10 (d, J=14.8Hz, 1H), 2.75 (m, 1H), 7.18 (t, J=7.8 Hz, 1H), 7.56 (d, J=7.2 Hz, 1H),8.16 (d, J=4.8 Hz, 1H), 8.26 (s, 1H). mp>300° C. APCI MS (Positive Mode)m/z 571 [C₃₆H₅₀N₄O₂+H]⁺.

Example 13

(i) Preparation of 13b: (4a′R,6a′R,6b′S,8a′S,12a′S,14b′R)-Methyl4′,4′,6a′,6b′,11′,11′,14b′-heptamethyl-13′-vinyl-2′,4′,4a′,5′,6′,6a′,6b′,7′,8′,8a′,9′,10′,11′,12′,12a′,14′,14a′,14b′-octadecahydro-1′H-spiro[[1,3]dioxolane-2,3′-picene]-8a′-carboxylate

To a suspension of methyltriphenylphosphonium iodide (808 mg, 2.0 mmol)in benzene (30 mL) was added potassium tert-butoxide (224 mg, 2.0 mmol).The mixture was stirred at room temperature for 1 hour. A solution of 5c(600 mg, 1.11 mmol) in THF (5 mL) was added. The mixture was stirred atroom temperature for 1.5 hours. The reaction mixture was diluted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-15% EtOAc in hexanes) to afford thesub-title compound (490 mg, 82%).

¹H NMR (300 MHz, CDCl₃) δ 0.63 (s, 3H), 0.85 (s, 3H), 0.89 (s, 3H), 0.91(s, 3H), 0.94 (s, 3H), 0.97 (s, 3H), 0.99 (s, 3H), 1.11 (s, 3H),1.15-2.05 (m, 22H), 3.60 (s, 3H), 3.95 (m, 4H), 4.95 (m, 1H), 4.95 (d,J=9.3 Hz, 2H), 5.09 (d, J=16.2 Hz, 1H).

(ii) Preparation of 13c: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-vinyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 13b (600 mg, 1.11 mmol) and p-toluenesulfonic acidmonohydrate (211 mg, 1.11 mmol) in acetone (20 mL) and H₂O (2 mL) washeated at reflux for 4 hours. The reaction mixture was concentrated toremove acetone under reduced pressure and extracted with EtOAc (100 mL).The organic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-15% EtOAc in hexanes) to afford the sub-titlecompound (500 mg, 91%).

¹H NMR (300 MHz, CDCl₃) δ 0.72 (s, 1.8H), 0.81 (s, 1.2H), 0.89 (s, 3H),0.91 (s, 3H), 1.0-2.50 (m, 38H), 3.65 (s, 3H), 5.40-5.70 (m, 1H), 6.05(d, J=12.0 Hz, 0.4H), 6.53 (d, J=15.0 Hz, 0.6H).

(iii) Preparation of 13d: (4aS,6aS,6bR,8aR,12aR,14bS)-Methyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-vinyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.27 mL, 1.92 mmol) in THF (6 mL) wasadded n-butyllithium (0.81 mL, 2.5 M in hexanes, 2.02 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 13c(500 mg, 1.01 mmol) in THF (10 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (329 mg, 1.82 mmol) in THF (3 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-15% EtOAc in hexanes) to afford the sub-titlecompound (200 mg, 38%).

(iv) Preparation of 13e: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-vinyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 13d (200 mg, 0.38 mmol) and hydrazine (0.015 mL, 0.46 mmol)in EtOH (3 mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (180 mg, 89%). ESI MS (Positive Mode) m/z 534[C₃₄H₅₁N₃O₂+H]⁺.

(v) Preparation of 13:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-vinyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 13e (80 mg, 0.15 mmol) and lithium iodide (300 mg, 2.25mmol) in 2,6-lutidine (3 mL) was heated to 144° C. for 5 hours. Thereaction mixture was cooled to room temperature and neutralized with HCl(2 M) and extracted with CH₂Cl₂/i-PrOH (3:1). The organic phase wasdried (MgSO₄), filtered and concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) followed by preparative HPLC to provide the titlecompound (6 mg, 8%) as an off-white solid.

R_(f) 0.35 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.81 (s, 3H), 0.91 (s, 3H), 0.94 (s, 3H), 1.01(s, 3H), 1.17 (s, 3H), 1.22 (s, 3H), 1.25 (m, 3H), 1.30-2.30 (m, 19H),2.53 (d, J=15.0 Hz, 1H), 3.53 (m, 1H), 4.98 (d, J=12.3 Hz, 1H), 5.18 (d,J=19.2 Hz, 1H), 6.95 (dd, J=12.3, 19.2 Hz, 1H). mp>300° C. ESI MS(Positive Mode) m/z 520 [C₃₃H₄₉N₃O₂+H]⁺.

Example 14

(i) Preparation of 14b: (4aS,6aS,6bR,8aR,13aR,15bS)-Methyl12-amino-15-ethyl-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 13e (80 mg, 0.15 mmol) and 10% Pd(OH)₂/C (30 mg) in MeOH(10 mL) was stirred under a hydrogen balloon for 12 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (20 mL). The filtrate was concentrated to dryness and the residuepurified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (70 mg, 87%). ESI MS (Positive Mode) m/z536 [C₃₄H₅₃N₃O₂+H]⁺.

(ii) Preparation of 14:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-ethyl-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 14b (70 mg, 0.13 mmol) and lithium iodide (263 mg, 1.96mmol) in 2,6-lutidine (3 mL) was heated to 144° C. for 5 hours. Thereaction mixture was cooled to room temperature and neutralized with HCl(2 M) and extracted with CH₂Cl₂/i-PrOH (3:1). The organic phase wasdried (MgSO₄), filtered and concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) followed by preparative HPLC to provide the titlecompound (13 mg, 19%) as an off-white solid.

R_(f) 0.80 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.83 (s, 3H), 0.92 (s, 6H), 0.99 (s, 3H), 1.11(s, 3H), 1.21 (s, 3H), 1.29 (s, 3H), 1.30-2.30 (m, 25H), 2.44 (d, J=15.0Hz, 1H). mp>300° C. ESI MS (Positive Mode) m/z 522 [C₃₃H₅₁N₃O₂+H]⁺.

Example 15

(i) Preparation of 15b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl1443-aminophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (600 mg, 0.96 mmol), 3-aminophenylboronic acid (362 mg,2.66 mmol), Pd(PPh₃)₄ (104 mg, 0.09 mmol) and K₂CO₃ (353 mg, 2.56 mmol)in benzene (16 mL) and EtOH (5 mL) was heated at reflux for 12 hours.The reaction mixture was diluted with EtOAc (100 mL). The organic phasewas washed with brine then dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-40% EtOAc in hexanes) to afford the sub-title compound (400 mg, 65%).

¹H NMR (300 MHz, CDCl₃) δ 0.30 (s, 3H), 0.76 (s, 3H), 0.86 (s, 3H), 1.04(s, 3H), 1.06 (s, 3H), 1.08 (s, 3H), 1.19 (s, 3H), 1.20-2.50 (m, 24H),3.12 (m, 1H), 5.02 (m, 2H), 6.48 (m, 3H), 7.01 (m, 1H), 7.33 (m, 5H).

(ii) Preparation of 15c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl14-(3-aminophenyl)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.22 mL, 1.57 mmol) in THF (5 mL) wasadded n-butyllithium (0.64 mL, 2.5 M in hexanes, 1.61 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 15b(400 mg, 0.62 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (285 mg, 1.57 mmol) in THF (3 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (100 mg, 25%).

¹H NMR (300 MHz, CDCl₃) δ 0.32 (s, 3H), 0.76 (s, 3H), 0.86 (s, 3H),0.87-2.30 (m, 32H), 3.12 (m, 1H), 3.30 (s, 2H), 3.82 (m, 1H), 5.02 (m,2H), 6.48 (m, 3H), 7.01 (m, 1H), 7.33 (m, 5H).

(iii) Preparation of 15d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(3-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 15c (100 mg, 0.15 mmol) and hydrazine (0.020 mL) in EtOH (2mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-30% CMA in CH₂Cl₂) to afford thesub-title compound (70 mg, 69%). APCI MS (Positive Mode) m/z 675[C₄₄H₅₈N₄O₂+H]⁺.

(iv) Preparation of 15:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(3-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 15d (70 mg, 0.10 mmol) and 10% Pd(OH)₂/C (35 mg) in MeOH(12 mL) was stirred under a hydrogen balloon for 12 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-80% CMA in CH₂Cl₂) followed by preparative HPLC to provide the titlecompound (32 mg, 55%) as a brown solid.

R_(f) 0.70 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, DMSO-d₆) δ 0.30 (s, 3H), 0.78 (s, 3H), 0.85 (s, 3H),0.96 (s, 3H), 1.18 (s, 3H), 1.31 (s, 6H), 1.32-2.28 (m, 19H), 2.26 (d,J=14.7 Hz, 1H), 3.0 (m, 1H), 7.16 (m, 4H). mp>300° C. APCI MS (PositiveMode) m/z 585 [C₃₇H₅₂N₄O₂+H]⁺.

Example 16

(i) Preparation of 16b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(3-methoxyphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and 3-methoxyphenylboronic acid(92 mg, 0.60 mmol) in benzene (4 mL) and EtOH (1 mL) was added K₂CO₃(125 mg, 0.90 mmol). The mixture was sparged with nitrogen and thenPd(PPh₃)₄ (70 mg, 0.06 mmol) was added. The reaction mixture was heatedat 120° C. for 1 hour using microwave irradiation and then concentrated.The residue was dissolved in EtOAc (20 mL) and the solution was washedwith brine then dried (Na₂SO₄), filtered and concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-30% CMA in CH₂Cl₂) to afford the sub-title compound (179 mg, 86%).

¹H NMR (300 MHz, CDCl₃) δ 0.30-2.25 (m, 39H), 3.11 (m, 1H), 3.40 (s,2H), 3.82 (s, 3H), 5.08 (d, J=12.6 Hz, 1H), 5.20 (d, J=12.3 Hz, 1H),6.75 (m, 1H), 6.78 (s, 1H), 7.15 (t, J=7.8 Hz, 1H), 7.31-7.36 (m, 5H).APCI MS m/z 690 [C₄₅H₅₉N₃O₃+H]⁺.

(ii) Preparation of 16:(4aS,6aS,6bR,13aR)-12-Amino-15-(3-hydroxyphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 16b (179 mg, 0.25 mmol) and HBr (2 mL, 33% in AcOH) washeated at 120° C. for 1 hour. The reaction mixture was concentratedunder reduced pressure and the residue was purified by columnchromatography (silica, 0-80% CMA in CH₂Cl₂) to afford the titlecompound (20 mg, 13%).

R_(f) 0.30 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.34-2.37 (m, 41H), 3.15 (d, J=10.8 Hz, 1H),6.61-6.70 (m, 3H), 7.11 (t, J=7.8 Hz, 1H). APCI MS m/z 586[C₃₇H₅₁N₃O₃+H]⁺. m.p. 260-280° C. dec. HPLC (Method A) 92.4% (214 nm)t_(R)=15.6 min.

Example 17

(i) Preparation of 17b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl14-(3-fluorophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (300 mg, 0.48 mmol), 3-fluorophenylboronic acid (100mg, 0.72 mmol), Pd(PPh₃)₄ (55 mg, 0.048 mmol) and cesium carbonate (469mg, 1.44 mmol) in benzene (15 mL) and EtOH (5 mL) was heated at refluxfor 12 hours. The reaction mixture was diluted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% EtOAc in hexanes) to afford the sub-titlecompound (250 mg, 82%).

¹H NMR (300 MHz, CDCl₃) δ 0.25 (s, 3H), 0.78 (s, 3H), 0.83 (s, 3H), 1.05(s, 3H), 1.07 (s, 3H), 1.12 (s, 3H), 1.21 (s, 3H), 1.23-2.55 (m, 22H),3.01 (m, 1H), 5.04 (m, 2H), 6.87 (m, 3H), 7.15 (m, 1H), 7.34 (m, 5H).

(ii) Preparation of 17c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl11-cyano-14-(3-fluorophenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.17 mL, 1.22 mmol) in THF (6 mL) wasadded n-butyllithium (0.51 mL, 2.5 M in hexanes, 1.28 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 17b(408 mg, 0.64 mmol) in THF (4 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (232 mg, 1.28 mmol) in THF (3 mL) was added at −78° C.The reaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (280 mg, 66%).

¹H NMR (300 MHz, CDCl₃) δ 0.25 (s, 3H), 0.78 (s, 3H), 0.83 (s, 3H), 1.05(s, 3H), 1.07 (s, 3H), 1.12 (s, 3H), 1.22 (s, 3H), 1.23-2.30 (m, 20H),3.01 (m, 1H), 3.83 (m, 1H), 5.04 (m, 2H), 6.87 (m, 3H), 7.15 (m, 1H),7.34 (m, 5H).

(iii) Preparation of 17d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(3-fluorophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 17c (280 mg, 0.42 mmol) and hydrazine (0.053 mL) in EtOH (5mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (230 mg, 81%). ESI MS (Positive Mode) m/z 678[C₄₄H₅₆FN₃O₂+H]⁺.

(iv) Preparation of 17:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(3-fluorophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 17d (230 mg, 0.34 mmol) and 10% Pd(OH)₂/C (100 mg) in MeOH(6 mL) and EtOAc (6 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the titlecompound (110 mg, 55%) as a white solid.

R_(f) 0.75 (4:1 Methylene Chloride/Methanol).

¹H NMR (300 MHz, CD₃OD) δ 0.30 (s, 3H), 0.75 (s, 3H), 0.87 (s, 3H), 0.99(s, 3H), 1.16 (s, 3H), 1.19 (s, 3H), 1.25 (s, 3H), 1.26-2.25 (m, 19H),2.30 (d, J=14.7 Hz, 1H), 3.15 (m, 1H), 6.91 (t, J=8.4 Hz, 1H), 7.01 (m,2H), 7.28 (q, J=8.1 Hz, 1H). mp>300° C. APCI MS (Positive Mode) m/z 588[C₃₇H₅₀FN₃O₂+H]⁺.

Example 18

(i) Preparation of 18b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(3-(aminomethyl)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and 3-(aminomethyl)phenylboronicacid (169 mg, 0.90 mmol) in benzene (4 mL) and EtOH (1 mL) was addedK₂CO₃ (166 mg, 1.20 mmol). The mixture was sparged with nitrogen andthen Pd(PPh₃)₄ (70 mg, 0.06 mmol) was added. The reaction mixture washeated at 120° C. for 1 hour using microwave irradiation and thenconcentrated. The residue was dissolved in EtOAc (20 mL) and the organiclayer was washed with brine then dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) to afford the sub-titlecompound (170 mg, 82%).

¹H NMR (300 MHz, CDCl₃) δ 0.23-2.25 (m, 45H), 3.05 (m, 1H), 3.40 (s,1H), 3.82 (s, 2H), 5.06 (d, J=12.6 Hz, 1H), 5.23 (d, J=12.3 Hz, 1H),7.06-7.20 (m, 4H), 7.33-7.38 (m, 5H). APCI MS m/z 689 [C₄₅H₆₀N₄O₂+H]⁺.

(ii) Preparation of 18:(4aS,6aS,6bR,13aR)-12-Amino-15-(3-(aminomethyl)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 18b (160 mg, 0.23 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (100 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresidue was purified by column chromatography (silica, 0-70% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (48mg, 35%) as a solid.

R_(f) 0.13 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.27-2.34 (m, 41H), 2.99 (d, J=11.1 Hz, 1H),4.13 (s, 2H), 7.30-7.44 (m, 4H). APCI MS m/z 599 [C₃₈H₅₄N₄O₂+H]⁺. m.p.270-290° C. dec. HPLC (Method A)>99% (214 nm) t_(R)=12.4 min.

Example 19

(i) Preparation of 19b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(3-carbamoylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and 3-carbamoylphenylboronic acid(99 mg, 0.64 mmol) in benzene (4 mL) and EtOH (1 mL) was added K₂CO₃(125 mg, 0.90 mmol). The mixture was sparged with nitrogen and thenPd(PPh₃)₄ (69 mg, 0.06 mmol) was added. The reaction mixture was heatedat 120° C. for 1 hour using microwave irradiation and then concentrated.The residue was dissolved in EtOAc (20 mL) and the solution was washedwith brine then dried (Na₂SO₄), filtered and concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-40% CMA in CH₂Cl₂) to afford the sub-title compound (170 mg, 80%).APCI MS m/z 703 [C₄₅H₅₈N₄O₃+H]⁺.

(ii) Preparation of 19:(4aS,6aS,6bR,13aR)-12-Amino-15-(3-carbamoylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 19b (170 mg, 0.24 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (100 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) to afford the titlecompound (60 mg, 40%).

R_(f) 0.22 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.24-2.38 (m, 41H), 3.02 (d, J=11.4 Hz, 1H),7.42-7.45 (m, 2H), 7.73-7.76 (m, 2H). APCI MS m/z 613 [C₃₈H₅₂N₄O₃+H]⁺.m.p.>300° C. HPLC (Method A)>99% (214 nm) t_(R)=14.1 min.

Example 20

(i) Preparation of 20b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-methyl-3-nitrophenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (250 mg, 0.37 mmol) and 4-methyl-3-nitrophenylboronicacid (136 mg, 0.75 mmol) in benzene (4 mL) and EtOH (1 mL) was addedK₂CO₃ (156 mg, 1.13 mmol). The mixture was sparged with nitrogen andthen Pd(PPh₃)₄ (87 mg, 0.07 mmol) was added. The reaction mixture washeated at 120° C. for 1 hour using microwave irradiation. The solventwas removed under reduced pressure and then the residue was dissolved inEtOAc (20 mL). The solution was washed with brine then dried (Na₂SO₄),filtered and concentrated under reduced pressure. The residue waspurified by column chromatography (silica, 0-40% CMA in CH₂Cl₂) toafford the sub-title compound (186 mg, 69%).

¹H NMR (300 MHz, CD₃OD) δ 0.26-2.34 (m, 41H), 2.51 (s, 3H), 3.01 (m,1H), 5.05 (d, J=12.0 Hz, 1H), 5.23 (d, J=12.0 Hz, 1H), 7.22-7.37 (m,7H), 7.80 (s, 1H). APCI MS m/z 719 [C₄₅H₅₈N₄O₄+H]⁺.

(ii) Preparation of 20:(4aS,6aS,6bR,13aR)-12-Amino-15-(3-amino-4-methylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 20b (180 mg, 0.25 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (180 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (16 mg, 10%) as a solid.

R_(f) 0.23 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.31-2.27 (m, 41H), 2.36 (s, 3H), 2.99 (d,J=10.5 Hz, 1H), 7.14-7.32 (m, 3H). APCI MS m/z 599 [C₃₈H₅₄N₄O₂+H]⁺. m.p.220-240° C. dec. HPLC (Method A)>99% (214 nm) t_(R)=12.4 min.

Example 21

(i) Preparation of 21b: (4aS,6aS,6bR,12aR)-Benzyl11-cyano-14-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a mixture of IIj (300 mg, 0.46 mmol) and3-(dimethylamino)phenylboronic acid (458 mg, 2.78 mmol) in benzene (10mL) and EtOH (5 mL) was added K₂CO₃ (511 mg, 3.70 mmol). The mixture wassparged with nitrogen and then Pd(PPh₃)₄ (107 mg, 0.09 mmol) was added.The mixture was heated at 85° C. overnight and then concentrated underreduced pressure. The residue was dissolved in EtOAc (50 mL) and thesolution was washed with brine then dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-10% EtOAc in hexanes) to afford the sub-titlecompound (270 mg, 84%).

¹H NMR (300 MHz, CDCl₃) δ 0.29-2.30 (m, 41H), 2.95 (s, 6H), 3.13 (d,J=10.5 Hz, 1H), 3.81-3.91 (m, 1H), 5.07 (d, J=12.3 Hz, 1H), 5.17 (d,J=15.3 Hz, 1H), 6.51 (d, J=6.9 Hz, 1H), 6.51 (d, J=6.9 Hz, 1H),7.03-7.09 (m, 1H), 7.34-7.36 (m, 5H).

(ii) Preparation of 21c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 21b (266 mg, 0.38 mmol) and EtOH (4 mL) was addedhydrazine (60 μL, 1.93 mmol). The mixture was heated at 90° C. overnightand then concentrated under reduced pressure. The residue was purifiedby column chromatography (0-30% EtOAc in hexanes and 50% CMA in CH₂Cl₂)to afford the sub-title compound (191 mg, 70%).

¹H NMR (300 MHz, CD₃OD) δ 0.27-2.36 (m, 42H), 2.89 (s, 6H), 5.05 (m,1H), 5.22 (m, 1H), 6.49-7.06 (m, 4H), 7.35-7.7.36 (m, 5H). APCI MS m/z703 [C₄₆H₆₂N₄O₂+H]⁺.

(iii) Preparation of 21:(4aS,6aS,6bR,13aR)-12-Amino-15-(3-(dimethylamino)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 21c (180 mg, 0.25 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (90 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-40% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (36mg, 23%) as a solid.

R_(f) 0.44 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.27-2.43 (m, 41H), 3.04 (d, J=9.6 Hz, 1H),3.18 (s, 6H), 7.16-7.47 (m, 4H). APCI MS m/z 613 [C₃₉H₅₆N₄O₂+H]⁺.m.p.>300° C. HPLC (Method A) 98.1% (214 nm) t_(R)=12.0 min.

Example 22

(i) Preparation of 22b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(2-fluoropyridin-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (500 mg, 0.75 mmol), 2-fluoropyridin-4-ylboronic acid(210 mg, 1.50 mmol), Pd(PPh₃)₄ (80 mg, 0.075 mmol) and K₂CO₃ (310 mg,2.25 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and heatedto 120° C. by microwave for 1 hour. The reaction mixture was dilutedwith EtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (448 mg, 88%) as a brown solid. APCI MS (PositiveMode) m/z 679 [C₄₃H₅₅FN₄O₂+H]⁺.

(ii) Preparation of 22:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(2-(dimethylamino)pyridin-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 22b (150 mg, 0.22 mmol) and dimethylamine (4.0 mL, 2 M inMeOH, 8.0 mmol) was sealed and heated to 140° C. by microwave for 5hours. The mixture was concentrated to dryness. The residue and 10%Pd(OH)₂/C (50 mg) in MeOH (12 mL) was stirred under a hydrogen balloonfor 5 hours. The reaction mixture was filtered through a pad ofdiatomaceous earth and washed with CMA (25 mL). The filtrate wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) followed bypreparative HPLC to provide the title compound (25 mg, 19%) as anoff-white solid.

R_(f) 0.65 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.82 (s, 3H), 0.90 (s, 3H), 0.98(s, 3H), 1.22 (s, 3H), 1.31 (s, 6H), 1.35-2.20 (m, 24H), 2.28 (m, 1H),2.31 (d, J=14.8 Hz, 1H), 3.05 (m, 1H), 6.90 (s, 1H), 7.31 (s, 1H), 7.90(s, 1H). mp>300° C. APCI MS (Positive Mode) m/z 614 [C₃₈H₅₅N₅O₂+H]⁺.

Example 23

(i) Preparation of 23b: 2-Aminophenylboronic acid

To a solution of 2-nitrophenylboronic acid (500 mg, 2.99 mmol) and MeOH(10 mL) was added 10% Pd/C (250 mg). The mixture was stirred underhydrogen at atmospheric pressure for 2 hours. The catalyst was removedby filtration through diatomaceous earth and the filtrate wasconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the sub-titlecompound (182 mg, 44%).

¹H NMR (300 MHz, CD₃OD) δ 6.69-7.75 (m, 4H).

(ii) Preparation of 23c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(2-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (150 mg, 0.22 mmol) and 23b (77 mg, 0.56 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (124 mg, 0.90 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (52 mg, 0.04 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation and then concentrated under reduced pressure. Theresidue was dissolved in EtOAc (20 mL). The organic solution was washedwith brine then dried (Na₂SO₄), filtered and concentrated. The residuewas purified by column chromatography (silica, 0-40% CMA in CH₂Cl₂) toafford the sub-title compound (190 mg, 75%).

APCI MS m/z 675 [C₄₄H₅₈N₄O₂+H]⁺.

(iii) Preparation of 23:(4aS,6aS,6bR,13aR)-12-Amino-15-(2-aminophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 23c (180 mg, 0.26 mmol) and MeOH (20 mL) was flushed withnitrogen and then 10% Pd/C (150 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-100% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (17 mg, 10%) as a solid.

R_(f) 0.28 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.20-2.47 (m, 48H), δ 7.29 (d, J=7.2 Hz, 1H),δ 7.41-7.42 (m, 3H). APCI MS m/z 585 [C₃₇H₅₂N₄O₂+H]⁺. m.p. 210-230° C.dec. HPLC (Method A)>99% (214 nm) t_(R)=12.7 min.

Example 24

(i) Preparation of 24b: (4aS,6aS,6bR,10S,12aR)-Benzyl10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of oleanolic acid (1a, 6.0 g, 13.1 mmol), benzyl bromide (2.8g, 16.4 mmol), K₂CO₃ (2.7 g, 19.7 mmol) and DMF (120 mL) was stirred atroom temperature for 15 hours. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,30% EtOAc in hexanes) to provide the sub-title compound (6.6 g, 92%) asa white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.61 (s, 3H), 0.65-0.72 (m, 1H), 0.78 (s, 3H),0.87-1.00 (m, 10H), 1.05-1.46 (m, 12H), 1.49-1.72 (m, 12H), 1.81-2.05(m, 3H), 2.86-2.91 (m, 1H), 3.15-3.27 (m, 1H), 5.02-5.11 (m, 2H),5.43-5.45 (m, 1H), 7.31-7.49 (m, 5H).

(ii) Preparation of 24c: (4aS,6aS,6bR,10S,12aR)-Benzyl10-acetoxy-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of 24b (6.6 g, 12.1 mmol), DMAP (200 mg) and pyridine (65mL) was slowly added acetic anhydride (1.5 g, 14.5 mmol) at 0° C. undernitrogen. The mixture was allowed to slowly warm to room temperatureovernight and was quenched by pouring into one liter of H₂O. Theprecipitate was collected by filtration and dried in a vacuum oven at40° C. to provide the sub-title compound (6.6 g, 93%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.61 (s, 3H), 0.77-0.94 (m, 16H), 0.98-1.75(m, 21H), 1.84-2.02 (m, 3H), 2.04 (s, 3H), 2.85-2.95 (m, 1H), 4.46-4.51(m, 1H), 5.01-5.08 (m, 2H), 5.27-5.29 (m, 1H), 7.29-7.35 (m, 5H).

(iii) Preparation of 24d: (4aS,6aS,6bR,10S,12aR)-Benzyl10-acetoxy-14-chloro-2,2,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a suspension of 24c (3.0 g, 5.13 mmol) and pyridine (65 mL) at 0° C.was added chlorine gas which was generated by addition of nitric acid tosodium chloride. After 20 min at 0° C., the mixture was poured into H₂Oand the solid was collected by filtration. The crude material waspurified by column chromatography (silica, 20% diethyl ether in hexanes)to provide the sub-title compound (1.3 g, 40%).

¹H NMR (500 MHz, CDCl₃) δ 0.82-0.91 (m, 12H), 1.00 (s, 3H), 1.12-1.79(m, 22H), 1.88-2.33 (m, 9H), 3.63-3.74 (m, 1H), 4.45-4.52 (m, 1H),5.11-5.21 (m, 2H), 7.31-7.44 (m, 5H).

(iv) Preparation of 24e: (4aS,6aS,6bR,12aR)-Benzyl14-chloro-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of 24d (1.0 g, 1.62 mmol), KOH (360 mg, 4.86 mmol) and MeOH(70 mL) was heated at reflux for 96 hours. The solvent was removed andthe residue was partitioned between H₂O (50 mL) and CH₂Cl₂ (50 mL). Theorganic layer was separated then dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The crude product was dissolved inCH₂Cl₂ (40 mL) and the Dess-Martin reagent (900 mg, 2.1 mmol) was addedat room temperature. The mixture was stirred for 14 hours beforequenching with a solution of sodium thiosulfate (12.5 g) and saturatedNaHCO₃ (50 mL). After stifling 30 min, the layers were separated and theaqueous layer was extracted with CH₂Cl₂ (50 mL). The combined organiclayers were dried (Na₂SO₄), filtered and concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-25% EtOAc in hexanes) to provide the sub-title compound (785 mg, 100%)as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.64 (s, 3H), 0.89-1.18 (m, 21H), 1.22-2.07(m, 15H), 2.23-2.41 (m, 3H), 2.47-2.61 (m, 1H), 3.62-3.75 (m, 1H),5.05-5.16 (m, 2H), 7.29-7.41 (m, 5H).

(v) Preparation of 24f: (4aS,6aS,6bR,12aR)-Benzyl14-chloro-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A solution of diisopropylamine (0.2 mL, 1.46 mmol) and THF (7 mL) wascooled to −78° C. under nitrogen. A solution of n-butyllithium (2.5 M inhexanes, 0.65 mL, 1.63 mmol) was slowly added, maintaining the internaltemperature below −70° C. The solution was allowed to stir for 45 minand was then slowly added to a solution of 24e (500 mg, 0.86 mmol) andTHF (10 mL) at −78° C. under nitrogen. This solution was stirred for 30min after which time a suspension of p-toluenesulfonyl cyanide (310 mg,1.72 mmol) and THF (3 mL) was added over 15 min. The solution wasstirred for 30 min and then quenched by addition of saturated ammoniumchloride solution (10 mL) at −78° C. The mixture was allowed to warm toroom temperature overnight. The organic layer was separated and theaqueous layer was extracted with EtOAc. The combined organic layers weredried (Na₂SO₄) and filtered. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,0-25% EtOAc in hexanes) to provide the sub-title compound (350 mg, 67%).

¹H NMR (500 MHz, CD₃OD) δ 0.59 (s, 3H), 0.82-2.29 (m, 39H), 3.63-3.74(m, 1H), 5.03-5.15 (m, 2H), 7.31-2.7.45 (m, 5H).

(vi) Preparation of 24g: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-chloro-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A solution of 24f (350 mg, 0.58 mmol) and hydrazine (74 mg, 2.32 mmol)in EtOH (10 mL) was heated at reflux for 16 hours. The solvent andexcess hydrazine were removed under reduced pressure. The residue waspurified by column chromatography (silica, 0-50% CMA in CH₂Cl₂) toprovide the sub-title compound (175 mg, 50%).

¹H NMR (300 MHz, CDCl₃) δ 0.61 (s, 3H), 0.88 (s, 3H), 0.91 (s, 3H), 0.98(s, 3H), 1.09-2.06 (m, 27H), 2.23-2.35 (m, 3H), 3.66-3.78 (m, 1H),5.02-5.14 (m, 2H), 7.29-7.41 (m, 5H).

(vii) Preparation of 24:(4aS,6aS,6bR,13aR)-12-Amino-15-chloro-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A suspension of 24g (150 mg, 0.24 mmol), 10% Pd/C (100 mg) andEtOAc/MeOH (20% MeOH, 25 mL) was stirred under hydrogen at atmosphericpressure for 23 hours. The catalyst was removed by filtration throughdiatomaceous earth and the filtrate was concentrated under reducedpressure. The residue was purified by column chromatography (silica,50-100% CMA in CH₂Cl₂) to provide the title compound (48 mg, 38%) as anoff-white solid.

¹H NMR (300 MHz, CD₃OD) δ 0.89-1.01 (m, 12H), 1.12-1.30 (m, 12H),1.34-2.10 (m, 15H), 2.33-2.41 (m, 3H), 3.62-3.70 (m, 1H). APCI MS m/z528 [C₃₁H₄₆ClN₃O₂+H]⁺. HPLC>99% (area %), t_(R)=15.9 min.

Example 25

(i) Preparation of 25:(4aS,6aS,6bR,8aR,13aR,15bR)-12-Amino-15-bromo-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of II (75 mg, 0.11 mmol) and 10% Pd(OH)₂/C (75 mg) in EtOAc(10 mL) and MeOH (2 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) to afford the titlecompound (20 mg, 32%) as a brown solid.

R_(f) 0.40 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.91 (s, 6H), 0.93 (s, 3H), 1.0 (s, 3H), 1.12(s, 3H), 1.15 (s, 3H), 1.21 (s, 3H), 1.32-2.28 (m, 20H), 2.35 (d, J=14.4Hz, 1H), 2.53 (d, J=8.7 Hz, 2H), 3.53 (m, 1H). mp 268-270° C. ESI MS(Positive Mode) m/z 572 [C₃₁H₄₆BrN₃O₂+H]⁺.

Example 26

(i) Preparation of 26b: (4aS,6aS,6bR,8aR,12aR,14bS)-benzyl14-(furan-3-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (360 mg, 0.57 mmol), furan-3-ylboronic acid (194 mg,1.73 mmol), Pd(PPh₃)₄ (66 mg, 0.057 mmol) and cesium carbonate (564 mg,1.73 mmol) in benzene (15 mL) and EtOH (4 mL) was heated at reflux for12 hours. The reaction mixture was diluted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (340 mg, 97%).

¹H NMR (300 MHz, CDCl₃) δ 0.57 (s, 3H), 0.77 (s, 3H), 0.81 (s, 3H), 1.05(s, 3H), 1.07 (s, 3H), 1.12 (s, 3H), 1.22 (s, 3H), 1.23-2.55 (m, 22H),3.31 (m, 1H), 5.04 (m, 2H), 6.32 (s, 1H), 7.20 (s, 1H), 7.31 (m, 6H).

(ii) Preparation of 26c: (4aS,6aS,6bR,8aR,12aR,14bS)-benzyl11-cyano-14-(furan-3-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.12 mL, 0.84 mmol) in THF (5 mL) wasadded n-butyllithium (0.35 mL, 2.5 M in hexanes, 0.88 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 26b(270 mg, 0.44 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (203 mg, 1.12 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (150 mg, 54%).

¹H NMR (300 MHz, CDCl₃) δ 0.51 (s, 3H), 0.62 (s, 3H), 0.65 (s, 3H), 1.03(s, 3H), 1.04 (s, 3H), 1.07 (s, 3H), 1.16 (s, 3H), 1.23-2.55 (m, 21H),3.32 (m, 1H), 5.02 (m, 2H), 6.32 (s, 1H), 7.20 (s, 1H), 7.31 (m, 6H).

(iii) Preparation of 26d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(furan-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 26c (150 mg, 0.23 mmol) and hydrazine (0.030 mL) in EtOH (3mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (110 mg, 71%).

ESI MS (Positive Mode) m/z 650 [C₄₂H₅₅N₃O₃+H]⁺.

(iv) Preparation of 26:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(furan-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 26d (110 mg, 0.17 mmol) and 10% Pd(OH)₂/C (50 mg) in MeOH(6 mL) and EtOAc (6 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the titlecompound (20 mg, 21%) as a brown solid.

R_(f) 0.45 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, DMSO-d₆) δ 0.53 (s, 3H), 0.78 (s, 3H), 0.80 (s, 3H),0.84 (s, 3H), 1.05 (s, 3H), 1.16 (s, 6H), 1.26-2.25 (m, 19H), 2.25 (d,J=14.7 Hz, 1H), 6.48 (s, 1H), 7.48 (s, 1H), 7.59 (s, 1H). mp>300° C.APCI MS (Positive Mode) m/z 560 [C₃₅H₄₉N₃O₃+H]⁺.

Example 27

(i) Preparation of 27b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl14-(1-benzyl-1H-pyrazol-4-yl)-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylatecarboxylate

A mixture of IIi (300 mg, 0.48 mmol), 1-benzyl-1H-pyrazol-4-ylboronicacid (291 mg, 1.44 mmol), Pd(PPh₃)₄ (60 mg, 0.048 mmol) and K₂CO₃ (198mg, 1.44 mmol) in DMF (9 mL) was heated at 100° C. for 24 hours. Thereaction mixture was diluted with EtOAc (100 mL). The organic phase waswashed with brine then dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-20% EtOAc in hexanes) to afford the sub-title compound (280 mg, 83%).

¹H NMR (300 MHz, CDCl₃) δ 0.43 (s, 3H), 0.76 (s, 3H), 0.77 (s, 3H),0.78-2.53 (m, 37H), 3.32 (m, 1H), 5.02 (m, 4H), 7.15 (m, 4H), 7.31 (m,8H).

(ii) Preparation of 27c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl14-(1-benzyl-1H-pyrazol-4-yl)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.13 mL, 0.95 mmol) in THF (5 mL) wasadded n-butyllithium (0.4 mL, 2.5 M in hexanes, 1.0 mmol) at −78° C. Thesolution was stirred for 30 min. The LDA solution was added to 27b (350mg, 0.50 mmol) in THF (5 mL). The mixture was allowed to warm to −40° C.for 5 min and cooled to −78° C. A suspension of p-toluene sulfonylcyanide (181 mg, 1.0 mmol) in THF (2 mL) was added. The reaction mixturewas allowed to warm to −40° C. over 1.5 hours. The reaction was quenchedby saturated NH₄Cl (3 mL) and extracted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% EtOAc in hexanes) to afford the sub-titlecompound (160 mg, 44%).

(iii) Preparation of 27d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(1-benzyl-1H-pyrazol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 27c (160 mg, 0.22 mmol) and hydrazine (0.10 mL, 0.69 mmol)in EtOH (3 mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-40% CMA in CH₂Cl₂) to afford thesub-title compound (60 mg, 62%). APCI MS (Positive Mode) m/z 740[C₄₁H₅₅N₅O₂+H]⁺.

(iv) Preparation of 27:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrazol-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 27d (60 mg, 0.082 mmol) and 10% Pd(OH)₂/C (60 mg) in MeOH(12 mL) and EtOAc (2 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-80% CMA in CH₂Cl₂) to afford the titlecompound (8 mg, 18%) as a brown solid.

R_(f) 0.20 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD+CDCl₃) δ 0.52 (s, 3H), 0.77 (s, 3H), 0.88 (s,3H), 0.95 (s, 3H), 1.16 (s, 3H), 1.24 (s, 6H), 1.32-2.28 (m, 20H), 2.32(d, J=14.7 Hz, 1H), 7.57 (s, 1H), 7.82 (s, 1H). mp>300° C. APCI MS(Positive Mode) m/z 560 [C₃₄H₄₉N₅O₂+H]⁺.

Example 28

(i) Preparation of 28b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-14-(1-methyl-1H-pyrazol-4-yl)-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIj (100 mg, 0.15 mmol), 1-methyl-1H-pyrazol-4-ylboronicacid (75.0 mg, 0.60 mmol), Pd(PPh₃)₄ (34 mg, 0.030 mmol) and K₂CO₃ (184mg, 1.33 mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was sealed andheated to 120° C. by microwave for 1 hour. The reaction mixture wasdiluted with EtOAc (100 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-30% EtOAc in hexanes)to afford the sub-title compound (60 mg, 61%).

¹H NMR (300 MHz, CDCl₃) δ 0.54 (s, 3H), 0.76 (s, 3H), 0.80 (s, 3H),0.81-2.05 (m, 32H), 3.32 (m, 1H), 3.77 (s, 3H), 3.85 (m, 1H), 5.02 (m,2H), 7.10 (s, 1H), 7.33 (m, 6H).

(ii) Preparation of 28c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 28b (60 mg, 0.09 mmol) and hydrazine (0.05 mL) in EtOH (1.5mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-30% CMA in CH₂Cl₂) to afford thesub-title compound (33 mg, 54%). APCI MS (Positive Mode) m/z 664[C₄₂H₅₇N₅O₂+H]⁺.

(iii) Preparation of 28:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1-methyl-1H-pyrazol-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 28c (33 mg, 0.05 mmol) and 10% Pd(OH)₂/C (16 mg) in MeOH(12 mL) was stirred under a hydrogen balloon for 5 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) to afford the title compound (20 mg, 71%) as abrown solid.

R_(f) −0.64 (9:1 Methylene Chloride/Methanol).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.81 (s, 3H), 0.87 (s, 3H), 0.93(s, 3H), 1.15 (s, 3H), 1.23 (s, 3H), 1.24 (s, 3H), 1.25-2.18 (m, 19H),2.36 (d, J=14.8 Hz, 1H), 3.34 (m, 1H), 7.45 (s, 1H), 7.52 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 574 [C₃₅H₅₁N₅O₂+H]⁺.

Example 29

(i) Preparation of 29b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-4-yl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIi (400 mg, 0.64 mmol), pyridine-4-ylboronic acid (395 mg,3.21 mmol), Pd(PPh₃)₄ (222 mg, 0.19 mmol) and K₂CO₃ (443 mg, 3.21 mmol)in DMF (10 mL) was heated at 100° C. for 24 hours. The reaction mixturewas diluted with EtOAc (100 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-30% EtOAc in hexanes)to afford the sub-title compound (185 mg, 46%).

¹H NMR (300 MHz, CDCl₃) δ 0.32 (s, 3H), 0.75 (s, 3H), 0.89 (s, 3H), 1.02(s, 3H), 1.04 (s, 3H), 1.10 (s, 3H), 1.23 (s, 3H), 1.32-2.05 (m, 20H),2.30 (m, 1H), 2.45 (m, 1H), 2.98 (m, 1H), 5.10 (m, 2H), 7.10 (d, J=5.7Hz, 2H), 7.30 (m, 5H), 8.35 (d, J=5.7 Hz, 2H).

(ii) Preparation of 29c: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl1′-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyridin-4-yl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a solution of diisopropylamine (0.16 mL, 1.16 mmol) in THF (5 mL) wasadded n-butyllithium (0.48 mL, 2.5 M in hexanes, 1.2 mmol) at −78° C.The solution was stirred for 30 min. The LDA solution was added to 29b(326 mg, 0.52 mmol) in THF (5 mL). The mixture was allowed to warm to−40° C. for 5 min and cooled to −78° C. A suspension of p-toluenesulfonyl cyanide (188 mg, 1.0 mmol) in THF (2 mL) was added. Thereaction mixture was allowed to warm to −40° C. over 1.5 hours. Thereaction was quenched by saturated NH₄Cl (3 mL) and extracted with EtOAc(100 mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-30% EtOAc in hexanes) to afford the sub-titlecompound (171 mg, 51%).

¹H NMR (300 MHz, CDCl₃) δ 0.25 (s, 3H), 0.75 (s, 3H), 0.82 (s, 3H), 1.09(s, 3H), 1.13 (s, 3H), 1.15 (s, 3H), 1.17 (s, 3H), 1.18-2.25 (m, 20H),2.95 (m, 1H), 3.85 (m, 1H), 5.04 (m, 2H), 7.06 (m, 2H), 7.37 (m, 5H),8.44 (m, 2H).

(iii) Preparation of 29d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 29c (171 mg, 0.26 mmol) and hydrazine (0.025 mL, 0.80 mmol)in EtOH (3 mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (143 mg, 83%).

ESI MS (Positive Mode) m/z 661 [C₄₃H₅₆N₄O₂+H]⁺.

(iv) Preparation of 29:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-4-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 29d (143 mg, 0.21 mmol) and 10% Pd(OH)₂/C (50 mg) in MeOH(15 mL) was stirred under a hydrogen balloon for 12 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) to afford the title compound (85 mg, 72%) as abrown solid.

R_(f) 0.24 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.32 (s, 3H), 0.75 (s, 3H), 0.89 (s, 3H), 0.98(s, 3H), 1.10 (s, 3H), 1.19 (s, 3H), 1.25 (s, 3H), 1.32-2.28 (m, 20H),2.32 (d, J=14.7 Hz, 1H), 3.0 (m, 1H), 7.40 (d, J=5.7 Hz, 2H), 8.47 (d,J=5.7 Hz, 1H). mp>300° C. ESI MS (Positive Mode) m/z 571[C₃₆H₅₀N₄O₂+H]⁺.

Example 30

(i) Preparation of 30b: (4aS,6aS,6bR,8aR,12aR,14bS)-Benzyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-14-(pyrimidin-5-yl)-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

A mixture of IIj (200 mg, 0.30 mmol), pyrimidin-5-ylboronic acid (115mg, 0.91 mmol), Pd(PPh₃)₄ (34 mg, 0.030 mmol) and K₂CO₃ (184 mg, 1.33mmol) in benzene (3.5 mL) and EtOH (1.5 mL) was sealed and heated to120° C. by microwave for 1 hour. The reaction mixture was diluted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-40% EtOAc in hexanes) to afford thesub-title compound (90 mg, 40%).

(ii) Preparation of 30c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyrimidin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 30b (90 mg, 0.13 mmol) and hydrazine (0.04 mL) in EtOH (1.5mL) was heated at reflux overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-30% CMA in CH₂Cl₂) to afford thesub-title compound (65 mg, 71%).

APCI MS (Positive Mode) m/z 664 [C₄₂H₅₅N₅O₂+H]⁺.

(iii) Preparation of 30:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyrimidin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 30c (65 mg, 0.098 mmol) and 10% Pd(OH)₂/C (30 mg) in MeOH(10 mL) was stirred under a hydrogen balloon for 5 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-60% CMA in CH₂Cl₂) to afford the title compound (32 mg, 57%) as abrown solid.

R_(f) 0.50 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.31 (s, 3H), 0.80 (s, 3H), 0.89 (s, 3H), 1.02(s, 3H), 1.20 (s, 3H), 1.25 (s, 3H), 1.31 (s, 3H), 1.32-2.30 (m, 21H),2.33 (d, J=14.8 Hz, 1H), 2.88 (m, 1H), 8.78 (s, 1H), 9.04 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 572 [C₃₅H₄₉N₅O₂+H]⁺.

Example 31

(i) Preparation of 31b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(4-(benzyloxycarbonylamino)-3-fluorophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and4-(benzyloxycarbonylamino)-3-fluorophenylboronic acid (262 mg, 0.90mmol) in benzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (166 mg, 1.20mmol). The mixture was sparged with nitrogen and then Pd(PPh₃)₄ (69 mg,0.06 mmol) was added. The reaction mixture was heated at 120° C. for 1hour using microwave irradiation and then solvent was removed underreduced pressure. The residue was dissolved in EtOAc (20 mL) and washedwith brine. The organic layer was dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (190 mg, 76%).

APCI MS m/z 827 [C₅₂H₆₃N₄O₄+H]⁺.

(ii) Preparation of 31:(4aS,6aS,6bR,13aR)-12-Amino-15-(4-amino-3-fluorophenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 31b (190 mg, 0.22 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (300 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (35 mg, 21%) as as solid.

R_(f) 0.74 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.37-2.43 (m, 41H), 3.07 (dd, J=3.4, 6.5 Hz,1H), 6.99-7.10 (m, 3H). APCI MS m/z 603 [C₃₇H₅₁FN₄O₂+H]⁺. m.p. 180-200°C. dec. HPLC (Method A)>99% (214 nm) t_(R)=13.4 min.

Example 32

(i) Preparation of 32b: 4-((3-Bromofuran-2-yl)methyl)morpholine

To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol) andmorpholine (0.5 mL, 5.71 mmol) in CH₂Cl₂ (10 mL) was added sodiumtriacetoxyborohydride (1.2 g, 5.71 mmol). The mixture was stirred atroom temperature for 12 hours. The reaction mixture was diluted withEtOAc (100 mL). The organic phase was washed with aqueous NaHCO₃ andbrine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (670 mg, 89%).

¹H NMR (300 MHz, CDCl₃) δ 2.50 (s, 4H), 3.62 (s, 2H), 3.70 (m, 4H), 6.43(s, 1H), 7.35 (s, 1H).

(ii) Preparation of 32c: 2-(Morpholinomethyl)furan-3-ylboronic acid

To a solution of 32b (670 mg, 2.54 mmol) in THF (15 mL) was addedn-butyllithium (2.5 M in hexanes, 1.30 mL, 3.31 mmol) at −78° C. Themixture was stirred at −78° C. for 20 min. Triisopropyl borate (2.9 mL,12.7 mmol) was added. The reaction mixture was stirred at −78° C. for 1hour and quenched with HCl (2 M, 4 mL). The reaction mixture was stirredfor 5 min and neutralized by NaOH (2 M) and extracted with EtOAc (100mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (450 mg, 83%).

¹H NMR (300 MHz, CDCl₃) δ 2.40 (s, 4H), 3.65 (s, 2H), 3.68 (m, 4H), 6.52(s, 1H), 7.30 (s, 1H).

(iii) Preparation of 32d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (200 mg, 0.30 mmol), 32c (250 mg, 1.18 mmol), Pd(PPh₃)₄(35 mg, 0.030 mmol) and K₂CO₃ (207 mg, 1.50 mmol) in benzene (3.5 mL)and EtOH (1.5 mL) was sealed and heated to 120° C. by microwave for 1hour. The reaction mixture was diluted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (135 mg, 60%).

APCI MS (Positive Mode) m/z 749 [C₄₇H₆₄N₄O₄+H]⁺.

(iv) Preparation of 32:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 32d (135 mg, 0.18 mmol) and 10% Pd(OH)₂/C (100 mg) in MeOH(12 mL) and EtOAc (3 mL) was stirred under a hydrogen balloon for 6hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CMA) to afford the title compound(35 mg, 30%) as a brown solid.

R_(f) 0.63 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.81 (s, 3H), 0.89 (s, 3H), 0.99(s, 3H), 1.14 (s, 3H), 1.28 (s, 6H), 1.35-2.28 (m, 20H), 2.61 (m, 4H),2.90 (m, 1H), 3.53 (m, 6H), 6.33 (s, 1H), 7.45 (s, 1H). mp>300° C. dec.APCI MS (Positive Mode) m/z 659 [C₄₀H₅₈N₄O₄+H]⁺.

Example 33

(i) Preparation of 33b: 1-(Triisopropylsilyl)-1H-pyrrole-3-carbaldehyde

To a solution of 3-bromo-1-(triisopropylsilyl)-1H-pyrrole (2.0 g, 6.61mmol) in THF (30 mL) was added n-butyllithium (2.5 M in hexanes, 3.4 mL,8.60 mmol) at −78° C. The mixture was stirred at −78° C. for 10 min thenDMF (0.76 mL, 9.92 mmol) was added and the mixture was warmed to 0° C.over 1 hour. The reaction mixture was quenched by saturated NH₄Cl andextracted with EtOAc (200 mL). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-10% EtOAc in hexanes)to afford the sub-title compound (1.65 g, 100%).

¹H NMR (400 MHz, CDCl₃) δ 2.40 (m, 4H), 3.42 (s, 2H), 3.70 (m, 4H), 6.03(s, 1H), 6.09 (s, 1H), 6.74 (s, 1H), 8.30 (bs, 1H).

(ii) Preparation of 33c:4-Bromo-1-(triisopropylsilyl)-1H-pyrrole-3-carbaldehyde

To a solution of 33b (1.45 g, 5.8 mmol) in THF (40 mL) was addedN-bromosuccinimide (1.03 g, 5.8 mmol) at room temperature. The mixturewas stirred for 2 hours and quenched with H₂O (5 mL) and extracted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% EtOAc in hexanes) to afford thesub-title compound (1.4 g, 74%).

¹H NMR (400 MHz, CDCl₃) δ 1.15 (d, J=2.5 Hz, 18H), 1.58 (m, 3H), 6.70(s, 1H), 7.32 (s, 1H), 9.82 (s, 1H).

(iii) Preparation of 33d:4-((4-Bromo-1-(triisopropylsilyl)-1H-pyrrol-3-yl)methyl)morpholine

To a solution of 33c (1.5 g, 4.5 mmol) and morpholine (0.79 mL, 9.0mmol) in CH₂Cl₂ (30 mL) was added sodium triacetoxyborohydride (1.9 g,9.0 mmol). The mixture was stirred at room temperature for 12 hours. Thereaction mixture was diluted with EtOAc (200 mL). The organic phase waswashed with aqueous NaHCO₃ and brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% EtOAc in hexanes) to afford the sub-titlecompound (1.8 g, 91%).

¹H NMR (400 MHz, CDCl₃) δ 1.15 (d, J=2.5 Hz, 18H), 1.58 (m, 3H), 2.40(m, 4H), 3.40 (s, 2H), 3.72 (m, 4H), 6.61 (s, 1H), 6.65 (s, 1H).

(iv) Preparation of 33e:4-((4-(4,4,5,5-Tetramethyl-1,3-dioxolan-2-yl)-1-(triisopropylsilyl)-1H-pyrrol-3-yl)methyl)morpholine

To a solution of 33d (400 mg, 1.0 mmol) was added n-butyllithium (2.5 Min hexanes, 0.52 mmol, 1.3 mmol) at −78° C. The mixture was stirred for20 min and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.24mL, 1.2 mmol) was added. The mixture was stirred at −78° C. for 30 minand warmed to room temperature over 1 hour. The reaction was quenchedwith H₂O and extracted with EtOAc (100 mL). The organic phase was washedwith brine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was subjected to column chromatography (silica, 0-50% EtOAc inhexanes) to afford the still impure sub-title compound (448 mg, 100%)which was used without further purification.

(v) Preparation of 33f: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)-1-(triisopropylsilyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (200 mg, 0.30 mmol), 33e (500 mg, 1.06 mmol), Pd(PPh₃)₄(35 mg, 0.030 mmol) and cesium carbonate (390 mg, 1.20 mmol) in toluene(4.5 mL) and H₂O (0.3 mL) was sealed and heated to 140° C. by microwavefor 1 hour. The reaction mixture was diluted with EtOAc (100 mL). Theorganic phase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (176 mg, 65%).

(vi) Preparation of 33g: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 33f (176 mg, 0.19 mmol) in THF (3 mL) was addedtetrabutylammonium fluoride (0.38 mL, 1 M in THF, 0.38 mmol). Themixture was stirred at room temperature for 2 hours. The reactionmixture was diluted with EtOAc (100 mL). The organic phase was washedwith brine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (60 mg, 42%).

APCI MS (Positive Mode) m/z 748 [C₄₇H₆₅N₅O₃+H]⁺.

(vii) Preparation of 33:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)-1H-pyrrol-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 33g (60 mg, 0.08 mmol) and 10% Pd(OH)₂/C (30 mg) in MeOH(12 mL) and EtOAc (3 mL) was stirred under a hydrogen balloon for 6hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-70% CMA in CMA) to afford the title compound(35 mg, 67%) as a brown solid.

R_(f) 0.54 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.55 (s, 3H), 0.85 (s, 3H), 0.92 (s, 3H), 1.12(s, 3H), 1.15 (s, 3H), 1.38 (m, 6H), 1.39-2.20 (m, 21H), 2.26 (d, J=14.4Hz, 1H), 2.90 (m, 1H), 3.12 (m, 3H), 3.75 (m, 6H), 6.55 (s, 1H), 7.92(s, 1H). mp>300° C. dec. APCI MS (Positive Mode) m/z 658[C₄₀H₅₉N₅O₃+H]⁺.

Example 34

(i) Preparation of 34b: 4-((3-Bromothiophen-2-yl)methyl)morpholine

To a solution of 3-bromothiophene-2-carbaldehyde (500 mg, 2.62 mmol) andmorpholine (0.68 mL, 7.85 mmol) and CH₂Cl₂ (10 mL) was added sodiumtriacetoxyborohydride (1.11 g, 5.24 mmol). The mixture was stirred atroom temperature overnight. The resulting mixture was diluted with EtOAc(100 mL) and the organic layer was washed with brine then dried(Na₂SO₄), filtered and concentrated. The residue was purified by columnchromatography (silica, 0-5% MeOH in CH₂Cl₂) to afford the sub-titlecompound (580 mg, 84%).

¹H NMR (300 MHz, CDCl₃) δ 2.47-2.54 (m, 4H), 3.68-3.71 (m, 4H), 6.90 (d,J=5.2 Hz, 1H), 7.23 (d, J=5.2 Hz, 1H).

(ii) Preparation of 34c: 2-(Morpholinomethyl)thiophen-3-ylboronic acid

To a solution of 34b (500 mg, 1.90 mmol) in THF (10 mL) was addedn-butyllithium (1.14 mL, 2.86 mmol) at −78° C. After stifling for 20min, triisopropyl borate (1.99 mL, 8.69 mmol) was added at −78° C. Themixture was slowly warmed to room temperature and then quenched withaqueous HCl (2.0 M, 2 mL). The reaction mixture was neutralized with 2 MNaOH solution and extracted with EtOAc (3×10 mL) followed byi-PrOH/CH₂Cl₂ (1:2, 10 mL×2). The combined extracts were dried (Na₂SO₄),filtered and concentrated under reduced pressure. The residue waspurified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (220 mg, 51%).

¹H NMR (300 MHz, CDCl₃) δ 2.47-2.49 (m, 4H), 3.52-3.76 (m, 6H),6.91-6.98 (m, 1H), 7.22-7.24 (m, 1H).

(iii) Preparation of 34d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)thiophen-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (250 mg, 0.38 mmol) and 34c (220 mg, 0.96 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (182 mg, 1.32 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (87 mg, 0.075 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation. The solvent was concentrated under reducedpressure. The residue was taken up in EtOAc (20 mL) and washed withbrine. The organic layer was dried (Na₂SO₄), filtered and concentrated.The residue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (190 mg, 66%).

APCI MS m/z 765 [C₄₇H₆₄N₄O₃S+H]⁺.

(iv) Preparation of 34:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)thiophen-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 34d (190 mg, 0.24 mmol), EtOH (2 mL) and MeOH (8 mL) wasflushed with nitrogen and then 10% Pd/C (400 mg) was added. The mixturewas flushed with nitrogen followed by hydrogen. The mixture was stirredunder hydrogen at atmospheric pressure overnight. The catalyst wasremoved by filtration through diatomaceous earth and the filtrate wasconcentrated. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) followed by preparative HPLC to afford the titlecompound (18 mg, 11%) as a solid.

R_(f) 0.13 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.40 (s, 3H), 0.80 (s, 3H), 0.92 (s, 3H), 1.05(s, 3H), 1.10-2.67 (m, 32H), 3.30 (s, 3H), 3.97 (s, 4H), 4.57-4.62 (m,1H), 6.95 (d, J=4.95 Hz, 1H), 7.67 (d, J=4.95 Hz, 1H). APCI MS m/z 675[C₄₀H₅₈N₄O₃S+H]⁺. m.p. 280-300° C. dec. HPLC (Method A) 98.7% (214 nm)t_(R)=12.9 min.

Example 35

(i) Preparation of 35b: tert-Butyl4-((3-bromofuran-2-yl)methyl)piperazine-1-carboxylate

To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol) andtert-butyl piperazine-1-carboxylate (1.1 g, 5.71 mmol) in CH₂Cl₂ (10 mL)was added sodium triacetoxyborohydride (1.2 g, 5.71 mmol). The mixturewas stirred at room temperature for 12 hours. The reaction mixture wasdiluted with EtOAc (100 mL). The organic phase was washed with aqueousNaHCO₃ and brine then dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (910 mg, 92%).

¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.40 (m, 4H), 3.42 (m, 4H), 3.60(s, 2H), 6.35 (s, 1H), 7.35 (s, 1H).

(ii) Preparation of 35c:2-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)furan-3-ylboronic acid

To a solution of 35b (910 mg, 2.63 mmol) in THF (20 mL) was addedn-butyllithium (2.5 M in hexanes, 1.4 mL, 3.43 mmol) at −78° C. Themixture was stirred at −78° C. for 20 minutes. Triisopropyl borate (3.0mL, 13.2 mmol) was added. The reaction mixture was stirred at −78° C.for 1 hour and quenched with HCl (2 M, 4 mL). The reaction mixture wasstirred for 5 min then neutralized by NaOH (2 M) and extracted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (560 mg, 68%).

¹H NMR (300 MHz, CDCl₃) δ 1.42 (s, 9H), 2.40 (m, 4H), 3.65 (m, 4H), 3.68(s, 2H), 6.52 (s, 1H), 7.31 (s, 1H).

(iii) Preparation of 35d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(2-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)furan-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (300 mg, 0.45 mmol), 35c (418 mg, 1.35 mmol), Pd(PPh₃)₄(52 mg, 0.045 mmol) and K₂CO₃ (248 mg, 1.80 mmol) in benzene (3.5 mL)and EtOH (1.5 mL) was sealed and heated to 120° C. by microwave for 1hour. The reaction mixture was diluted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (400 mg, 100%).

APCI MS (Positive Mode) m/z 848 [C₅₂H₇₃N₅O₅+H]⁺.

(iv) Preparation of 35e: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(piperazin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 35d (400 mg, 0.45 mmol) in MeOH (3 mL) and CH₂Cl₂ (2mL) was added HCl (2.3 mL, 2 M in Et₂O, 4.6 mmol). The mixture wasstirred at room temperature for 12 hours. The reaction mixture wasconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (250 mg, 74%).

APCI MS (Positive Mode) m/z 748 [C₄₇H₆₅N₅O₃+H]⁺.

(v) Preparation of 35:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(piperazin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 35e (250 mg, 0.33 mmol) and 10% Pd(OH)₂/C (110 mg) in MeOH(15 mL) and EtOAc (5 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-10% MeOH in CMA) to afford the title compound(160 mg, 75%) as a brown sold.

R_(f) 0.20 (80:28:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.78 (s, 3H), 0.86 (s, 3H), 0.94(s, 3H), 1.12 (s, 3H), 1.21 (s, 3H), 1.24 (s, 3H), 1.35-2.18 (m, 19H),2.36 (d, J=14.8 Hz, 1H), 2.73 (m, 4H), 3.0 (m, 1H), 3.18 (m, 4H), 3.52(m, 1H), 3.90 (m, 1H), 6.33 (s, 1H), 7.44 (s, 1H). mp>300° C. APCI MS(Positive Mode) m/z 658 [C₄₀H₅₉N₅O₃+H]⁺.

Example 36

(i) Preparation of 36b: 1-((3-Bromofuran-2-yl)methyl)-4-methylpiperazine

To a solution of 3-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol),1-methylpiperazine dihydrochloride (986 mg, 5.71 mmol) and triethylamine(1.5 mL, 11.4 mmol) in CH₂Cl₂ (10 mL) was added sodiumtriacetoxyborohydride (1.2 g, 5.71 mmol). The mixture was stirred atroom temperature for 12 hours. The reaction mixture was diluted withEtOAc (100 mL). The organic phase was washed with aqueous NaHCO₃ andbrine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (570 mg, 78%).

¹H NMR (300 MHz, CDCl₃) δ 2.25 (s, 3H), 2.40 (m, 4H), 2.60 (m, 4H), 3.60(s, 2H), 6.35 (s, 1H), 7.35 (s, 1H).

(ii) Preparation of 36c:2-((4-Methylpiperazin-1-yl)methyl)furan-3-ylboronic acid

To a solution of 36b (570 mg, 2.20 mmol) in THF (15 mL) was addedn-butyllithium (2.5 M in hexanes, 1.1 mL, 2.80 mmol) at −78° C. Themixture was stirred at −78° C. for 20 minutes. Triisopropyl borate (2.5mL, 11.0 mmol) was added. The reaction mixture was stirred at −78° C.for 1 hour and quenched with HCl (2 M, 4 mL). The reaction mixture wasstirred for 5 min then neutralized by NaOH (2 M) and extracted withEtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-15% MeOH in CH₂Cl₂) to afford thesub-title compound (200 mg, 41%).

¹H NMR (300 MHz, CD₃OD) δ 2.32 (s, 3H), 2.40 (m, 4H), 2.80 (m, 4H), 3.95(s, 2H), 6.46 (s, 1H), 7.38 (s, 1H).

(iii) Preparation of 36d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-((4-methylpiperazin-1-yl)methyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (200 mg, 0.30 mmol), 36c (200 mg, 0.89 mmol), Pd(PPh₃)₄(35 mg, 0.030 mmol) and K₂CO₃ (166 mg, 1.20 mmol) in benzene (3.5 mL)and EtOH (1.5 mL) was sealed and heated to 120° C. by microwave for 1hour. The reaction mixture was diluted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the sub-titlecompound (200 mg, 87%).

APCI MS (Positive Mode) m/z 762 [C₄₈H₆₇N₅O₃+H]⁺.

(iv) Preparation of 36:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-((4-methylpiperazin-1-yl)methyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 36d (200 mg, 0.26 mmol) and 10% Pd(OH)₂/C (100 mg) in MeOH(15 mL) and EtOAc (5 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-70% CMA in CH₂Cl₂) to afford the titlecompound (22 mg, 13%) as a brown solid.

R_(f) 0.30 (80:28:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.78 (s, 3H), 0.86 (s, 3H), 0.99(s, 3H), 1.12 (s, 3H), 1.28 (s, 6H), 1.35-2.18 (m, 16H), 2.28 (d, J=14.8Hz, 1H), 2.29 (m, 4H), 2.40 (m, 9H), 2.90 (m, 1H), 3.52 (m, 2H), 3.80(m, 1H), 6.33 (s, 1H), 7.47 (s, 1H). mp>300° C. APCI MS (Positive Mode)m/z 672 [C₄₁H₆₁N₅O₃+H]⁺.

Example 37

(i) Preparation of 37b: 1-((3-Bromofuran-2-yl)methyl)pyrrolidine

To a solution of 3-bromofuran-2-carbaldehyde (1.5 g, 8.57 mmol) andpyrrolidine (1.42 mL, 17.14 mmol) and CH₂Cl₂ (30 mL) was added sodiumtriacetoxyborohydride (3.63 g, 17.14 mmol). The mixture was stirred atroom temperature for 5 hours. The resulting mixture was diluted withEtOAc (300 mL) and the organic layer was washed with saturated NaHCO₃and brine then dried (Na₂SO₄), filtered and concentrated. The residuewas purified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (1.26 g, 65%).

¹H NMR (400 MHz, CDCl₃) δ 1.74-1.80 (m, 4H), 2.56-2.59 (m, 4H), 3.67 (s,2H), 6.37 (d, J=2.0 Hz, 1H), 7.34 (d, J=2.0 Hz, 1H).

(ii) Preparation of 37c: 2-(Pyrrolidin-1-ylmethyl)furan-3-ylboronic acid

To a solution of 37b (1.29 g, 5.60 mmol) in THF (20 mL) was addedn-butyllithium (4.26 mL, 10.65 mmol) at −78° C. After stirring for 20min, triisopropylborate (1.99 mL, 8.69 mmol) was added at −78° C. Themixture was slowly warmed to room temperature and then quenched withH₃PO₄ (85% in H₂O, 3 mL). The reaction mixture was neutralized with 2 NNaOH solution and extracted with EtOAc (3×10 mL) followed byi-PrOH/CH₂Cl₂ (2:1, 3×10 mL). The combined extracts were dried (Na₂SO₄),filtered and concentrated under reduced pressure. The residue waspurified by column chromatography (silica, 0-50% CMA in CH₂Cl₂) toafford the sub-title compound (515 mg, 47%).

¹H NMR (400 MHz, CD₃OD) δ 2.04-2.07 (m, 4H), 3.15-3.18 (m, 4H),3.29-3.31 (m, 2H), 4.19 (s, 2H), 6.41 (d, J=1.6 Hz, 1H), 7.32 (d, J=1.6Hz, 1H).

(iii) Preparation of 37d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(pyrrolidin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (500 mg, 0.75 mmol) and 37c (441 mg, 2.26 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (416 mg, 3.02 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (174 mg, 0.15 mmol)was added. The reaction mixture was heated at 130° C. for 1 hour usingmicrowave irradiation. The solvent was concentrated under reducedpressure. The residue was dissolved in EtOAc (40 mL) and washed withbrine (3×15 mL). The organic layer was dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂ followed by 30% CMA in CH₂Cl₂) to afford thesub-title compound (260 mg, 47%).

¹H NMR (400 MHz, CD₃OD) δ 0.51-2.66 (m, 50H), 3.05-3.08 (m, 1H),3.61-3.87 (m, 3H), 5.0 (d, J=12.20 Hz, 1H), 5.21 (d, J=12.20 Hz, 1H),6.23 (d, J=1.56 Hz, 1H), 7.31-7.52 (m, 7H). APCI MS m/z 733[C₄₇H₆₄N₄O₃+H]⁺.

(iv) Preparation of 37:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(pyrrolidin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 37d (260 mg, 0.35 mmol), EtOAc (5 mL) and MeOH (10 mL) wasflushed with nitrogen and then 10% Pd/C (130 mg) was added. The mixturewas flushed with nitrogen followed by hydrogen. The mixture was stirredunder hydrogen at atmospheric pressure overnight. The catalyst wasremoved by filtration through diatomaceous earth and the filtrate wasconcentrated. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) to afford the title compound (85 mg, 37%).

R_(f) 0.24 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.59 (s, 3H), 0.85 (s, 3H), 0.89 (s, 3H),1.04-2.32 (m, 36H), 2.75-2.79 (m, 1H), 3.11-3.15 (m, 2H), 3.48 (s, 2H),4.08-4.19 (m, 2H), 6.38 (d, J=1.5 Hz, 1H), 7.60 (d, J=1.5 Hz, 1H). APCIMS m/z 643 [C₄₀H₅₈N₄O₃+H]⁺. m.p. 260-280° C. dec. HPLC (Method A) 98.4%(214 nm) t_(R)=13.1 min

Example 38

(i) Preparation of 38b: 1-((4-Bromofuran-2-yl)methyl)pyrrolidine

To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol) andpyrrolidine (0.47 mL, 5.74 mmol) and CH₂Cl₂ (10 mL) was added sodiumtriacetoxyborohydride (1.2 g, 5.71 mmol). The mixture was stirred atroom temperature overnight. The resulting mixture was diluted with EtOAc(100 mL) and the organic layer was washed with brine then dried(Na₂SO₄), filtered and concentrated. The residue was purified by columnchromatography (silica, 0-5% MeOH in CH₂Cl₂) to afford the sub-titlecompound (400 mg, 61%).

¹H NMR (300 MHz, CDCl₃) δ 1.77-1.82 (m, 4H), 2.53-2.58 (m, 4H), 3.62 (s,2H), 6.25 (s, 1H), 7.35 (s, 1H).

(ii) Preparation of 38c: 5-(Pyrrolidin-1-ylmethyl)furan-3-ylboronic acid

To a solution of 38b (400 mg, 1.73 mmol) in THF (10 mL) was addedn-butyllithium (0.90 mL, 2.26 mmol) at −78° C. After stifling for 20min, triisopropylborate (1.99 mL, 8.69 mmol) was added at −78° C. Themixture was slowly warmed to room temperature and then quenched withphosphoric acid. The reaction mixture was neutralized with 2 M NaOHsolution and extracted with EtOAc (3×10 mL) followed by i-PrOH/CH₂Cl₂(1:2, 2×10 mL). The combined extracts were dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-100% CMA in CH₂Cl₂) to afford the sub-titlecompound (197 mg, 58%).

¹H NMR (300 MHz, CD₃OD) δ 1.84 (d, J=3.9 Hz, 4H), 2.74 (m, 4H), 3.81 (d,J=4.2 Hz, 2H), 6.46 (d, J=4.2 Hz, 1H), 7.62 (d, J=3.9 Hz, 1H).

(iii) Preparation of 38d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(pyrrolidin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and 38c (176 mg, 0.96 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (166 mg, 1.20 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (69 mg, 0.06 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation. The solvent was concentrated under reducedpressure. The residue was taken up in EtOAc (20 mL) and washed withbrine. The organic layer was dried (Na₂SO₄), filtered and concentrated.The residue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (200 mg, 90%).

¹H NMR (300 MHz, CD₃OD) δ 0.59-3.69 (m, 52H), 5.03 (d, J=12.0 Hz, 1H),5.21 (d, J=12.3 Hz, 1H), 6.22 (s, 1H), 7.21 (s, 1H), 7.58-7.67 (m, 5H).APCI MS m/z 733 [C₄₇H₆₄N₄O₃+H]⁺.

(iv) Preparation of 38:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(pyrrolidin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 38d (200 mg, 0.27 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (300 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-80% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (16mg, 9%) as a solid.

R_(f) 0.30 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.62-2.19 (m, 46H), 2.41 (d, J=15.0 Hz, 2H),3.53 (m, 2H), 4.45 (s, 2H), 6.64 (s, 1H), 7.57 (s, 1H). APCI MS m/z 643[C₄₀H₅₈N₄O₃+H]⁺. m.p. 220-240° C. dec. HPLC (Method A) 98.6% (214 nm)t_(R)=12.3 min.

Example 39

(i) Preparation of 39b: tert-Butyl4-((4-bromofuran-2-yl)methyl)piperazine-1-carboxylate

To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol) andCH₂Cl₂ (10 mL) was added tert-butyl piperazine-1-carboxylate (1.06 g,5.74 mmol) and sodium triacetoxyborohydride (1.2 g, 5.74 mmol). Themixture was stirred at room temperature overnight. The resultant mixturewas diluted with EtOAc (100 mL), washed with brine then dried (Na₂SO₄),filtered and concentrated under reduced pressure. The residue waspurified by column chromatography (silica, 0-5% MeOH in CH₂Cl₂) toafford the sub-title compound (900 mg, 91%).

¹H NMR (300 MHz, CDCl₃) δ 1.45 (s, 9H), 2.39-2.42 (m, 4H), 3.42-3.45 (m,4H), 3.52 (s, 2H), 6.27 (s, 1H), 7.38 (s, 1H).

(ii) Preparation of 39c:5-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)furan-3-ylboronic acid

To a solution of 39b (900 mg, 2.60 mmol) and THF (10 mL) was addedn-butyllithium (1.35 mL, 3.39 mmol) at −78° C. After stifling for 10min, triisopropylborate (2.99 mL, 13.04 mmol) was added at −78° C. Themixture was slowly warmed to room temperature and quenched withphosphoric acid. The reaction mixture was neutralized with 2 M NaOHsolution and extracted with EtOAc (3×10 mL). The extracts were dried(Na₂SO₄), filtered and concentrated under reduced pressure. The residuewas purified by column chromatography (silica, 0-5% CMA in CH₂Cl₂) toafford the sub-title compound (180 mg, 58%).

¹H NMR (300 MHz, CDCl₃) δ 1.44 (s, 9H), 3.44 (m, 4H), 3.56 (s, 2H), 3.73(m, 4H), 6.39 (s, 1H), 7.87 (s, 1H).

(iii) Preparation of 39d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(5-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)furan-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-Octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (300 mg, 0.45 mmol) and 39c (420 mg, 1.35 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (249 mg, 1.80 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (105 mg, 0.09 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation. The solvent was removed under reduced pressureand the residue was dissolved in EtOAc (20 mL). The solution was washedwith brine then dried (Na₂SO₄), filtered and concentrated. The residuewas purified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (260 mg, 68%).

¹H NMR (300 MHz, CDCl₃) δ 0.57-2.41 (m, 65H), 3.42-3.48 (m, 6H), 5.04(d, J=14.7 Hz, 1H), 5.14 (d, J=12.3 Hz, 1H), 6.18 (s, 1H), 7.21 (s, 1H),7.33-7.35 (m, 5H). APCI MS m/z 848 [C₅₂H₇₃N₅O₅+H]⁺.

(iv) Preparation of 39e: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(piperazin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 39d (260 mg, 0.30 mmol) and MeOH (2 mL) was added HCl(1.0 M in diethyl ether, 10 mL, 10.0 mmol). After stifling overnight atroom temperature, the reaction mixture was concentrated. Purification ofthe residue by column chromatography (silica, 0-80% CMA in CH₂Cl₂)afforded the sub-title compound (190 mg, 83%).

¹H NMR (300 MHz, CD₃OD) δ 0.57-2.37 (m, 42H), 2.44 (s, 4H), 2.80 (s,4H), 3.43 (s, 2H), 4.98-5.03 (m, 1H), 5.15-5.21 (m, 1H), 6.20 (s, 1H),7.21 (s, 1H), 7.36-7.37 (m, 5H). APCI MS m/z 748 [C₄₇H₆₅N₅O₃+H]⁺.

(v) Preparation of 39:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(piperazin-1-ylmethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 39e (190 mg, 0.25 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (190 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-70% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (34 mg, 20%) as a solid.

R_(f) 0.10 (32:17:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.60-2.44 (m, 42H), 3.14 (s, 4H), 3.39 (s,4H), 4.06 (s, 2H), 6.64 (s, 1H), 7.48 (s, 1H).

APCI MS m/z 658 [C₄₀H₅₉N₅O₃+H]⁺. m.p. 220-240° C. dec. HPLC (MethodA)>99% (214 nm) t_(R)=13.7 min.

Example 40

(i) Preparation of 40b: 4-((4-Bromofuran-2-yl)methyl)morpholine

To a solution of 4-bromofuran-2-carbaldehyde (500 mg, 2.85 mmol) andmorpholine (0.5 mL, 5.71 mmol) in CH₂Cl₂ (10 mL) was added sodiumtriacetoxyborohydride (1.2 g, 5.71 mmol). The mixture was stirred atroom temperature for 12 hours. The reaction mixture was diluted withEtOAc (100 mL). The organic phase was washed with aqueous NaHCO₃ andbrine then dried (MgSO₄), filtered and concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (600 mg, 86%).

¹H NMR (300 MHz, CDCl₃) δ 2.42 (m, 4H), 3.62 (s, 2H), 3.72 (m, 4H), 6.27(s, 1H), 7.32 (s, 1H).

(ii) Preparation of 40c: 5-(Morpholinomethyl)furan-3-ylboronic acid

To a solution of 40b (300 mg, 1.20 mmol) in THF (6 mL) was addedn-butyllithium (2.5 M in hexanes, 0.64 mL, 1.60 mmol) at −78° C. Themixture was stirred at −78° C. for 20 min. Triisopropyl borate (1.3 mL,6.0 mmol) was added. The reaction mixture was stirred at −78° C. for 1hour and quenched with HCl (2 M, 4 mL). The reaction mixture was stirredfor 5 min and neutralized by NaOH (2 M) and extracted with EtOAc (100mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (120 mg, 47%).

¹H NMR (300 MHz, CDCl₃) δ 2.40 (s, 4H), 3.65 (s, 2H), 3.68 (m, 4H), 6.58(s, 1H), 7.95 (s, 1H).

(iii) Preparation of 40d: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (100 mg, 0.18 mmol), 40c (120 mg, 0.56 mmol), Pd(PPh₃)₄(20 mg, 0.018 mmol) and K₂CO₃ (100 mg, 0.72 mmol) in benzene (3.5 mL)and EtOH (1.5 mL) was sealed and heated to 120° C. by microwave for 1hour. The reaction mixture was diluted with EtOAc (100 mL). The organicphase was washed with brine then dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (120 mg, 89%).

APCI MS (Positive Mode) m/z 749 [C₄₇H₆₄N₄O₄+H]⁺.

(iv) Preparation of 40:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(5-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 40d (120 mg, 0.16 mmol) and 10% Pd(OH)₂/C (40 mg) in MeOH(13 mL) and EtOAc (2 mL) was stirred under a hydrogen balloon for 6hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) followed by preparativeHPLC to provide the title compound (25 mg, 24%) as an off-white solid.

R_(f) 0.25 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.60 (s, 3H), 0.84 (s, 3H), 0.89 (s, 3H), 0.94(s, 3H), 1.22 (s, 3H), 1.25 (s, 3H), 1.30 (s, 3H), 1.35-2.28 (m, 20H),2.38 (d, J=15.0 Hz, 1H), 3.85 (m, 3H), 4.44 (s, 2H), 6.86 (s, 1H), 7.60(s, 1H). mp>300° C. APCI MS (Positive Mode) m/z 659 [C₄₀H₅₈N₄O₄+H]⁺.

Example 41

(i) Preparation of 41b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(6-fluoropyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (400 mg, 2.85 mmol), 6-fluoropyridin-3-ylboronic acid(400 mg, 2.85 mmol), Pd(PPh₃)₄ (105 mg, 0.090 mmol) and K₂CO₃ (600 mg,4.34 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and heatedto 120° C. by microwave for 1 hour. The reaction mixture was dilutedwith EtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (500 mg, 81%) as a brown solid.

APCI MS (Positive Mode) m/z 679 [C₄₃H₅₅FN₄O₂+H]⁺.

(ii) Preparation of 41:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-aminopyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 41b (100 mg, 0.14 mmol) and hydrazine (0.5 mL) in MeOH (4mL) was sealed and heated to 140° C. by microwave for 5 hours. Themixture was concentrated to dryness. The residue was dissolved in MeOH(20 mL), transferred to a Parr hydrogenation flask and Raney Ni (70 mg)was added. The mixture was placed on a Parr shaker under 40 psi ofhydrogen for 12 hours. The mixture was filtered through a pad ofdiatomaceous earth and the filter cake washed with CMA. The filtrate wasconcentrated to dryness and the residue dissolved in MeOH (10 mL),transferred to a Parr hydrogenation flask and 10% Pd(OH)₂/C (70 mg) wasadded and the mixture placed on a Parr Shaker under 30 psi of hydrogenfor 12 hours. The reaction mixture was filtered through a pad ofdiatomaceous earth and washed with CMA (25 mL). The filtrate wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-60% CMA in CH₂Cl₂) to afford thetitle compound (8 mg, 7%) as a brown solid.

R_(f) 0.30 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.82 (s, 3H), 0.90 (s, 3H), 0.98(s, 3H), 1.22 (s, 3H), 1.31 (s, 6H), 1.35-2.30 (m, 21H), 6.61 (d, J=8.1Hz, 1H), 7.47 (d, J=8.1 Hz, 1H), 7.80 (s, 1H). mp>300° C. dec. APCI MS(Positive Mode) m/z 586 [C₃₆H₅₁N₅O₂+H]⁺.

Example 42

(i) Preparation of 42b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(6-methoxypyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (200 mg, 0.30 mmol), 6-methoxypyridin-3-ylboronic acid(138 mg, 0.90 mmol), Pd(PPh₃)₄ (34 mg, 0.030 mmol) and K₂CO₃ (208 mg,1.50 mmol) in benzene (4.0 mL) and EtOH (1.0 mL) was sealed and heatedto 120° C. by microwave for 1 hour. The reaction mixture was dilutedwith EtOAc (100 mL). The organic phase was washed with brine then dried(MgSO₄), filtered and concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (200 mg, 96%) as a brown solid.

APCI MS (Positive Mode) m/z 691 [C₄₄H₅₈N₄O₃+H]⁺.

(ii) Preparation of 42:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-methoxypyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

To a solution of 42b (107 mg, 0.15 mmol) in CH₂Cl₂ (2 mL) was addedboron tribromide (1 M in THF, 0.93 mL, 0.93 mmol) at −78° C. The mixturewas warmed to 0° C. for 6 hours. The reaction mixture was quenched withMeOH (2 mL) and concentrated to dryness under reduced pressure. Theresidue was purified by column chromatography (silica, 0-4% CMA inCH₂Cl₂) to afford the title compound (34 mg, 38%).

R_(f) 0.75 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.32 (s, 3H), 0.78 (s, 3H), 0.88 (s, 3H), 1.0(s, 3H), 1.16 (s, 3H), 1.25 (s, 3H), 1.28 (s, 3H), 1.30-2.25 (m, 19H),2.32 (d, J=14.7 Hz, 1H), 3.04 (m, 1H), 3.89 (s, 3H), 6.78 (d, J=8.7 Hz,1H), 7.62 (d, J=10.2, 1H), 8.02 (s, 1H). mp>300° C. APCI MS (PositiveMode) m/z 601 [C₃₇H₅₂N₄O₃+H]⁺.

Example 43

(i) Preparation of 43b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(methylamino)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 41b (100 mg, 0.14 mmol) and methylamine (1.5 mL, 1 M inTHF, 1.5 mmol) in i-PrOH (2 mL) was sealed and heated to 160° C. bymicrowave for 5 hours. The mixture was concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-30% CMA inCH₂Cl₂) to afford the sub-title compound (41 mg, 42%) as a brown solid.

APCI MS (Positive Mode) m/z 690 [C₄₄H₅₉N₅O₂+H]⁺.

(ii) Preparation of 43:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(methylamino)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 43b (32 mg, 0.046 mmol) and 10% Pd(OH)₂/C (32 mg) in MeOH(20 mL) was stirred under a hydrogen balloon for 5 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-70% CMA in CH₂Cl₂) followed by preparative HPLC to provide the titlecompound (10 mg, 37%) as an off-white solid.

R_(f) 0.60 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.82 (s, 3H), 0.90 (s, 3H), 0.98(s, 3H), 1.22 (s, 3H), 1.28 (s, 3H), 1.31 (s, 3H), 1.35-2.30 (m, 21H),3.02 (m, 1H), 3.03 (s, 3H), 7.05 (d, J=9.3 Hz, 1H), 7.83 (s, 1H), 7.90(d, J=8.4 Hz, 1H). mp>300° C. APCI MS (Positive Mode) m/z 600[C₃₇H₅₃N₅O₂+H]⁺.

Example 44

(i) Preparation of 44b: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-15-(6-(dimethylamino)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of 41b (150 mg, 0.22 mmol) and dimethylamine (2.0 mL, 1 M inMeOH, 2.0 mmol) in MeOH (1 mL) was sealed and heated to 140° C. bymicrowave for 7 hours. The mixture was concentrated to dryness. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (88 mg, 58%) as a brown solid.

APCI MS (Positive Mode) m/z 690 [C₄₅H₆₁N₅O₂+H]⁺.

(ii) Preparation of 44:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-15-(6-(dimethylamino)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 44b (88 mg, 0.12 mmol) and 10% Pd(OH)₂/C (40 mg) in MeOH(15 mL) was stirred under a hydrogen balloon for 12 hours. The reactionmixture was filtered through a pad of diatomaceous earth and washed withCMA (25 mL). The filtrate was concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-40% CMA in CH₂Cl₂) to afford the title compound (35 mg, 26%) as anoff-white solid.

R_(f) 0.75 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.38 (s, 3H), 0.78 (s, 3H), 0.87 (s, 3H), 0.99(s, 3H), 1.15 (s, 3H), 1.25 (s, 3H), 1.26 (s, 3H), 1.35-2.30 (m, 19H),2.33 (d, J=14.4 Hz, 1H), 3.06 (s, 6H), 3.07 (m, 1H), 6.66 (d, J=8.7 Hz,1H), 7.50 (d, J=8.7 Hz, 1H), 7.94 (s, 1H). mp>300° C. dec. APCI MS(Positive Mode) m/z 614 [C₃₈H₅₅N₅O₂+H]⁺.

Example 45

(i) Preparation of 45b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(pyrrolidin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 41b (150 mg, 0.22 mmol) in MeOH (4 mL) was addedpyrrolidine (1 mL, 12.09 mmol). The mixture was heated at 140° C. for 5hours using microwave irradiation. The solvent was removed under reducedpressure and the residue was dissolved in EtOAc (20 mL). The solutionwas washed with brine then dried (Na₂SO₄), filtered and concentrated toafford the sub-title compound (162 mg, 100%) which was used withoutfurther purification.

APCI MS m/z 730 [C₄₇H₆₃N₅O₂+H]⁺.

(ii) Preparation of 45:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(pyrrolidin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 45b (162 mg, 0.22 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (200 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-40% CMA inCH₂Cl₂) to afford the title compound (52 mg, 37%).

R_(f) 0.48 (43:6:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.39 (s, 3H), 0.78 (s, 3H), 0.87-2.27 (m,38H), 2.38 (d, J=14.6 Hz, 1H), 3.08 (m, 1H), 3.43 (m, 4H), 6.50 (d,J=8.7 Hz, 1H), 7.51 (dd, J=2.1 Hz, 2.9 Hz, 1H), 7.90 (s, 1H). m.p.280-300° C. dec. APCI MS m/z 640 [C₄₀H₅₇N₅O₂+H]⁺.

HPLC (Method A)>99% (214 nm) t_(R)=12.2 min.

Example 46

(i) Preparation of 46b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(isoquinolin-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.30 mmol) and isoquinolin-5-ylboronic acid(156 mg, 0.90 mmol) in benzene (4 mL) and EtOH (1 mL) was added K₂CO₃(166 mg, 1.20 mmol). The mixture was sparged with nitrogen and thenPd(PPh₃)₄ (70 mg, 0.06 mmol) was added. The reaction mixture was heatedat 120° C. for 1 hour using microwave irradiation and then concentrated.The residue was dissolved in EtOAc (20 mL) and washed with brine. Theorganic solution was dried (Na₂SO₄), filtered and concentrated underreduced pressure. The residue was purified by column chromatography(silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (180 mg,84%).

APCI MS m/z 711 [C₄₇H₅₈N₄O₂+H]⁺.

(ii) Preparation of 46:(4aS,6aS,6bR,13aR)-12-Amino-15-(isoquinolin-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 46b (180 mg, 0.25 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (220 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-80% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (13 mg, 8%) as a solid.

R_(f) 0.73 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ −0.17-2.33 (m, 41H), 2.95 (d, J=11.1 Hz, 1H),7.94-7.99 (m, 1H), 8.13 (d, J=6.3 Hz, 1H), 8.19 (d, J=7.2 Hz, 1H), 8.32(d, J=8.4 Hz, 1H), 8.54 (d, J=6.3 Hz, 1H), 9.61 (s, 1H). APCI MS m/z 621[C₄₀H₅₂N₄O₂+H]⁺. m.p.>300° C. HPLC (Method A) 93.4% (214 nm) t_(R)=11.6min.

Example 47

(i) Preparation of 47b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1,2,3,4-tetrahydroisoquinolin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 46b (150 mg, 0.21 mmol) and THF (5 mL) was addedlithium triethylborohydride (1.26 mL, 1.26 mmol, 1.0 M in THF). Themixture was heated at 60° C. overnight and then cooled to roomtemperature. The mixture was taken up in EtOAc (40 mL) and washed withbrine (3×15 mL). The organic layer was dried (Na₂SO₄), filtered andconcentrated. The residue was purified by column chromatography (silica,0-80% CMA in CH₂Cl₂) to afford the sub-title compound (40 mg, 26%).

APCI MS m/z 715 [C₄₇H₆₂N₄O₂+H]⁺.

(ii) Preparation of 47:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(1,2,3,4-tetrahydroisoquinolin-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 47b (40 mg, 0.055 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (50 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-80% CMA inCH₂Cl₂) to afford the title compound (19 mg, 56%).

R_(f) 0.28 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.24 (s, 3H), 0.75-2.36 (m, 38H), 2.86-3.44(m, 5H), 4.10-4.20 (m, 2H), 6.98 (d, J=7.42 Hz, 1H), 7.19 (d, J=7.42 Hz,1H), 7.40 (d, J=7.42 Hz, 1H). APCI MS m/z 625 [C₄₀H₅₆N₄O₂+H]⁺. m.p.>300°C. dec. HPLC (Method A) 99.1% (214 nm) t_(R)=12.4 min

Example 48

(i) Preparation of 48b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(1H-indol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.302 mmol) and 1H-indol-4-ylboronic acid(146 mg, 0.906 mmol) in toluene (4.5 mL) and H₂O (0.5 mL) was addedcesium carbonate (393 mg, 1.208 mmol). The mixture was sparged withnitrogen and then Pd(PPh₃)₄ (70 mg, 0.060 mmol) was added. The reactionmixture was heated at 120° C. for 1 hour using microwave irradiation.The solvent was concentrated under reduced pressure. The residue wastaken up in EtOAc (20 mL) and washed with brine (3×15 mL). The organiclayer was dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (180 mg, 85%).

APCI MS m/z 699 [C₄₆H₅₈N₄O₂+H]⁺.

(ii) Preparation of 48:(4aS,6aS,6bR,13aR)-12-Amino-15-(1H-indol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 48b (180 mg, 0.26 mmol), EtOAc (2 mL) and MeOH (8 mL) wasflushed with nitrogen and then 10% Pd/C (280 mg) was added. The mixturewas flushed with nitrogen followed by hydrogen. The mixture was stirredunder hydrogen at atmospheric pressure overnight. The catalyst wasremoved by filtration through diatomaceous earth and the filtrate wasconcentrated. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) followed by preparative HPLC to afford the titlecompound (29 mg, 18%) as a solid.

R_(f) 0.30 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ −0.08 (s, 3H), 0.22 (s, 3H), 0.54 (s, 3H),0.78-2.56 (m, 32H), 6.28 (s, 1H), 7.01-7.24 (m, 5H). APCI MS m/z 609[C₃₉H₅₂N₄O₂+H]⁺. m.p. 280-300° C. dec. HPLC (Method A) 99.3% (214 nm)t_(R)=16.6 min

Example 49

(i) Preparation of 49b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (200 mg, 0.302 mmol) and 1H-indol-5-ylboronic acid(146 mg, 0.91 mmol) in toluene (4.5 mL) and H₂O (0.5 mL) was addedcesium carbonate (393 mg, 1.21 mmol). The mixture was sparged withnitrogen and then Pd(PPh₃)₄ (70 mg, 0.06 mmol) was added. The reactionmixture was heated at 120° C. for 1 hour using microwave irradiation.The solvent was concentrated under reduced pressure. The residue wastaken up in EtOAc (20 mL) and washed with brine (3×15 mL). The organiclayer was dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (silica, 0-10% CMA in CH₂Cl₂) toafford the sub-title compound (178 mg, 84%).

APCI MS m/z 699 [C₄₆H₅₈N₄O₂+H]⁺.

(ii) Preparation of 49:(4aS,6aS,6bR,13aR)-12-Amino-15-(1H-indol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 49b (175 mg, 0.25 mmol), EtOAc (5 mL) and MeOH (10 mL) wasflushed with nitrogen and then 10% Pd/C (175 mg) was added. The mixturewas flushed with nitrogen followed by hydrogen. The mixture was stirredunder hydrogen at atmospheric pressure overnight. The catalyst wasremoved by filtration through diatomaceous earth and the filtrate wasconcentrated. The residue was purified by column chromatography (silica,0-6% MeOH in CH₂Cl₂) followed by preparative HPLC to afford the titlecompound (18 mg, 12%) as a solid.

R_(f) 0.30 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.10 (s, 3H), 0.70 (s, 3H), 0.89-2.43 (m,35H), 3.21-3.25 (m, 1H), 6.38-6.39 (m, 1H), 7.04 (s, 1H), 7.18 (s, 1H),7.32 (d, J=8.26 Hz, 1H), 7.39 (s, 1H). APCI MS m/z 609 [C₃₉H₅₂N₄O₂+H]⁺.m.p. 230-250° C. dec. HPLC (Method A) 96.2% (214 nm) t_(R)=17.1 min

Example 50

(i) Preparation of 50b: tert-Butyl4-(4-bromophenyl)piperazine-1-carboxylate

To a slurry of 1-(4-bromophenyl)piperazine hydrochloride (3.0 g, 10.8mmol), DMAP (135 mg, 1.1 mmol) and triethylamine (4.5 mL, 32.4 mmol) inCH₃CN (25 mL) was added di-tert-butyl dicarbonate (2.6 g, 11.9 mmol).The mixture was stirred at room temperature overnight. Water (25 mL) wasadded to the resultant mixture and stirred for 30 min. The resultantsolid was collected by filtration. The solid was washed with H₂O anddried in a vacuum oven at 40° C. to afford the sub-title compound (3.2g, 87%).

¹H NMR ((300 MHz, DMSO-d₆) δ 1.48 (s, 9H), 3.15 (m, 4H), 3.54 (m, 4H),6.95 (d, J=9.0 Hz, 2H), 7.37 (d, J=9.0 Hz, 2H).

(ii) Preparation of 50c:4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)phenylboronic acid

To a solution of 50b (500 mg, 1.46 mmol) and THF (10 mL) was addedn-butyllithium (0.76 mL of 2.5 M in hexanes, 1.90 mmol) at −78° C. Afterstirring for 30 min, triisopropylborate (1.68 mL, 7.33 mmol) was addedat −78° C. The mixture was slowly warmed to room temperature andquenched with phosphoric acid. The reaction mixture was neutralized withsaturated NaHCO₃ and extracted with EtOAc (10 mL×3). The extracts weredried (Na₂SO₄), filtered and concentrated under reduced pressure. Theresidue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the sub-title compound (180 mg, 58%).

¹H NMR (300 MHz, CD₃OD) δ −0.50 (s, 9H), 0.93-0.96 (m, 4H), 1.15-1.19(m, 4H), 4.86-4.89 (m, 4H).

(iii) Preparation of 50d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (130 mg, 0.19 mmol) and 50c (180 mg, 0.58 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (108 mg, 0.78 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (45 mg, 0.06 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation and then concentrated under reduced pressure. Theresidue was dissolved in EtOAc (20 mL) and the solution was washed withbrine then dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (silica, 0-5% MeOH in CH₂Cl₂) toafford the sub-title compound (100 mg, 60%).

¹H NMR (300 MHz, CD₃OD) δ 0.28-3.59 (m, 59H), 5.18 (d, 2H), 6.70-7.07(m, 9H).

APCI MS m/z 844 [C₅₃H₇₃N₅O₄+H]⁺.

(iv) Preparation of 50e: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(piperazin-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 50d (220 mg, 0.26 mmol) in MeOH (3 mL) was added HCl(1.0 M in diethyl ether, 6 mL, 6.0 mmol). The mixture was stirred atroom temperature for 62 hours and then concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) to afford the sub-title compound (65 mg, 33%).

¹H NMR (300 MHz, CD₃OD) δ 0.27-2.34 (m, 42H), 2.98-3.06 (m, 8H), 5.02(d, J=12.3 Hz, 1H), 5.22 (d, J=12.0 Hz, 1H), 6.78 (d, J=8.7 Hz, 2H),6.99 (d, J=8.7 Hz, 2H), 7.39 (m, 5H). APCI MS m/z 744 [C₄₈H₆₅N₅O₂+H]⁺.

(v) Preparation of 50:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(piperazin-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 50e (65 mg, 0.087 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (90 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-70% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (11mg, 20%) as a solid.

R_(f) 0.10 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.28-2.42 (m, 41H), 3.10 (d, J=10.2 Hz, 1H),3.37 (m, 8H), 7.03 (d, J=9.0 Hz, 2H), 7.24 (d, J=7.8 Hz, 2H). APCI MSm/z 654 [C₄₁H₅₉N₅O₂+H]⁺. m.p. 270-290° C. dec. HPLC (Method A)>99% (214nm) t_(R)=13.2 min.

Example 51

(i) Preparation of 51b: (4aS,6aS,6bR,12aR)-Benzyl11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-14-(4-morpholinophenyl)-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylate

To a mixture of IIj (300 mg, 0.46 mmol) and 4-morpholinophenylboronicacid (575 mg, 2.78 mmol) in benzene (10 mL) and EtOH (5 mL) was addedK₂CO₃ (511 mg, 3.70 mmol). The mixture was sparged with nitrogen andthen Pd(PPh₃)₄ (107 mg, 0.09 mmol) was added. The mixture was heated at85° C. overnight and concentrated under reduced pressure. The residuewas dissolved in EtOAc (50 mL) and the solution was washed with brinethen dried (Na₂SO₄), filtered and concentrated. The residue was purifiedby column chromatography (silica, 0-30% EtOAc in hexanes) to afford thesub-title compound (200 mg, 59%).

¹H NMR (300 MHz, CDCl₃) δ 0.28-2.29 (m, 43H), 3.09-3.10 (m, 4H),3.85-3.86 (m, 4H), 5.05 (m, 1H), 5.17 (m, 1H), 6.75-6.78 (m, 2H),7.02-7.06 (m, 2H), 7.25-7.36 (m, 5H).

(ii) Preparation of 51c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-morpholinophenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 51b (196 mg, 0.26 mmol) and EtOH (5 mL) was addedhydrazine (42 μL, 1.34 mmol). The mixture was heated at 90° C. for 60hours and then concentrated under reduced pressure. The residue waspurified by column chromatography (silica, 0-20% CMA in CH₂Cl₂) toprovide the sub-title compound (109 mg, 55%).

¹H NMR (300 MHz, CDCl₃) δ 0.30-2.24 (m, 43H), 3.07-3.12 (m, 4H),3.40-3.47 (m, 2H), 3.85-3.88 (m, 4H), 5.10 (d, J=12.3 Hz, 1H), 5.22 (d,J=12.6 Hz, 1H), 6.78 (d, J=8.7 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H),7.36-7.39 (m, 5H). APCI MS m/z 745 [C₄₈H₆₄N₄O₃+H]⁺.

(iii) Preparation of 51:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-morpholinophenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 51c (108 mg, 0.14 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (55 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-10% MeOH inCH₂Cl₂) to afford the title compound (26 mg, 27%).

R_(f) 0.35 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.30-2.37 (m, 42H), 3.10 (m, 4H), 3.84 (m,4H), 6.95 (d, J=7.8 Hz, 2H), 7.16 (d, J=7.5 Hz, 2H). APCI MS m/z 655[C₄₁H₅₈N₄O₃+H]⁺. m.p. 250-270° C. dec. HPLC (Method A)>99% (214 nm)t_(R)=13.3 min.

Example 52

(i) Preparation of 52b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(4-formylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of II (250 mg, 0.37 mmol), 4-formylphenylboronic acid (170mg, 1.13 mmol), benzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (208 mg,1.50 mmol). The mixture was sparged with nitrogen and then Pd(PPh₃)₄ (87mg, 0.07 mmol) was added. The reaction mixture was heated at 120° C. for1 hour using microwave irradiation and then concentrated under reducedpressure. The residue was dissolved in EtOAc (20 mL) and the solutionwas washed with brine. The organic layer was dried (Na₂SO₄), filteredand concentrated. The residue was purified by column chromatography(silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (224 mg,86%).

APCI MS m/z 688 [C₄₅H₅₇N₃O₃+H]⁺.

(ii) Preparation of 52c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 52b (220 mg, 0.32 mmol), morpholine (56 μL, 0.64 mmol)and CH₂Cl₂ (10 mL) was added sodium triacetoxyborohydride (136 mg, 0.64mmol). The mixture was stirred at room temperature overnight. Theresultant mixture was diluted with EtOAc (100 mL) and the solution waswashed with brine then dried (Na₂SO₄), filtered and concentrated underreduced pressure. The residue was purified by column chromatography(silica, 0-20% MeOH in CH₂Cl₂) to afford the sub-title compound (80 mg,33%).

¹H NMR (300 MHz, CD₃OD) δ 0.28-2.24 (m, 41H), 2.39-2.42 (m, 4H), 3.03(m, 1H), 3.45 (s, 2H), 3.69-3.70 (m, 4H), 5.03 (d, J=12.6 Hz, 1H), 5.23(d, J=12.3 Hz, 1H), 7.15-7.18 (m, 4H), 7.37 (s, 5H).

(iii) Preparation of 52:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(morpholinomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 52c (75 mg, 0.09 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (100 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by preparative HPLC to afford the titlecompound (8 mg, 12%).

R_(f) 0.75 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.25-2.39 (m, 45H), 2.99 (m, 1H), 3.73 (s,2H), 4.02 (s, 2H), 4.38 (s, 2H), 7.47-7.52 (m, 4H). APCI MS m/z 669[C₄₂H₆₀N₄O₃+H]⁺. m.p.>300° C. dec. HPLC (Method A)>99% (214 nm)t_(R)=12.1 min.

Example 53

(i) Preparation of 53b: 1-(4-Bromophenyl)-4-methylpiperazine

A mixture of 1-(4-bromophenyl)piperazine hydrochloride (1.0 g, 3.61mmol), formaldehyde (37% aqueous, 3 mL, 39.71 mmol), AcOH (0.23 mL, 3.97mmol) and MeOH (30 mL) was heated at 80° C. under nitrogen. After 1.5hours, the mixture was cooled to 0° C. in an ice bath. Methylenechloride (5 mL) was added followed by a slow addition of sodiumborohydride (1.91 g, 50.54 mmol) under nitrogen. The mixture was stirredfor 1 hour and then poured into a seperatory funnel containing asaturated solution of NH₄Cl (25 mL) and CH₂Cl₂ (50 mL). The layers wereseparated and the aqueous layer was extracted with CH₂Cl₂ (2×50 mL). Theextracts were dried (Na₂SO₄), filtered and concentrated to to give thesub-title compound (900 mg, 97%) which was used without furtherpurification.

¹H NMR ((300 MHz, CDCl₃) δ 2.34 (s, 3H), 2.55 (m, 4H), 3.17 (m, 4H),6.79 (d, J=9.0 Hz, 2H), 7.32 (d, J=9.0 Hz, 2H).

(ii) Preparation of 53c: 4-(4-Methylpiperazin-1-yl)phenylboronic acid

To a solution of 53b (390 mg, 1.52 mmol) and THF (10 mL) was addedn-butyllithium (0.79 mL of 2.5 M in hexanes, 1.98 mmol) at −78° C. andstirred for 10 min. Triisopropylborate (1.75 mL, 7.64 mmol) was added at−78° C. The mixture was slowly warmed to room temperature and thenquenched with phosphoric acid. The reaction mixture was neutralized withsaturated NaHCO₃ solution and extracted with EtOAc (3×10 mL). Thesolution was dried (Na₂SO₄), filtered and concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (180 mg, 58%).

¹H NMR ((300 MHz, CD₃OD) δ 2.32 (s, 3H), 2.55 (m, 4H), 3.03 (s, 1H),3.21 (m, 3H), 6.69-7.59 (m, 4H).

(iii) Preparation of 53d: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(4-methylpiperazin-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a mixture of II (160 mg, 0.24 mmol) and 53c (159 mg, 0.72 mmol) inbenzene (4 mL) and EtOH (1 mL) was added K₂CO₃ (133 mg, 0.96 mmol). Themixture was sparged with nitrogen and then Pd(PPh₃)₄ (55 mg, 0.04 mmol)was added. The reaction mixture was heated at 120° C. for 1 hour usingmicrowave irradiation and then concentrated. The residue was dissolvedin EtOAc (20 mL) and the solution was washed with brine then dried(Na₂SO₄), filtered and concentrated under reduced pressure. The residuewas purified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (80 mg, 44%).

¹H NMR (300 MHz, CDCl₃) δ 0.29-3.46 (m, 53H), 5.06 (d, J=12.6 Hz, 1H),5.22 (d, J=12.6 Hz, 1H), 6.80 (d, J=8.4 Hz, 2H), 7.05 (d, J=8.7 Hz, 2H),7.35-7.44 (m, 5H). APCI MS m/z 758 [C₄₉H₆₇N₅O₂+H]⁺.

(iv) Preparation of 53:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(4-(4-methylpiperazin-1-yl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 53d (75 mg, 0.09 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (70 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-40% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (6 mg,5%) as a solid.

R_(f) 0.37 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.28-2.42 (m, 34H), 2.97 (s, 3H), 3.12-3.77(m, 8H), 3.37 (s, 8H), 7.02 (d, J=8.7 Hz, 2H), 7.24 (d, J=8.1 Hz, 2H).APCI MS m/z 668 [C₄₂H₆₁N₅O₂+H]⁺. m.p.>300° C. dec. HPLC (Method A)>99%(214 nm) t_(R)=13.9 min.

Example 54

(i) Preparation of 54:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-morpholinopyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 41b (100 mg, 0.14 mmol) and morpholine (0.5 mL) in i-PrOH(2 mL) was sealed and heated to 160° C. by microwave for 2 hours. Themixture was concentrated to dryness. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the sub-title compound (10 mg, 11%) as an off-whitesolid.

R_(f) 0.75 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.46 (s, 3H), 0.82 (s, 3H), 0.90 (s, 3H), 0.99(s, 3H), 1.22 (s, 3H), 1.25 (s, 3H), 1.29 (s, 3H), 1.35-2.30 (m, 19H),2.37 (d, J=14.4 Hz, 1H), 3.01 (s, 1H), 3.62 (m, 4H), 3.84 (m, 4H), 7.34(d, J=9.6 Hz, 1H), 8.00 (s, 2H). mp>300° C. APCI MS (Positive Mode) m/z656 [C₄₀H₅₇N₅O₃+H]⁺.

Example 55

(i) Preparation of 55:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(piperazin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

To 41b (150 mg, 0.22 mmol) in MeOH (5 mL) was added piperazine (1.38 g,20.89 mmol). The reaction mixture was heated at 140° C. for 5 hours andthen at 150° C. for an additional 5 hours using microwave irradiation.The solvent was concentrated and then the residue was dissolved inEtOAc/i-PrOH/CH₂Cl₂ (1:1:1, 30 mL). The solution was washed with brine,dried (Na₂SO₄), filtered and concentrated. The residue was purified bycolumn chromatography (silica, 0-40% CMA in CH₂Cl₂) followed bypreparative HPLC to afford the title compound (15 mg, 10%) as a solid.

R_(f) 0.10 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.40-2.43 (m, 41H), 2.98 (m, 1H), 3.39-3.41(m, 4H), 3.85-3.87 (m, 4H), 7.23 (d, J=9.0 Hz, 1H), 7.89 (d, J=3.4 Hz,1H), 8.12 (s, 1H). APCI MS m/z 655 [C₄₀H₅₈N₆O₂+H]⁺. m.p. 240-260° dec.HPLC (Method A) 96.2% (214 nm) t_(R)=10.7 min.

Example 56

(i) Preparation of 56b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 41b (210 mg, 0.30 mmol) in MeOH (4 mL) was added1-methylpiperazine dihydrochloride (1.5 g, 8.67 mmol). The reactionmixture was heated at 140° C. for 10 hours using microwave irradiationand then concentrated. The residue was dissolved in EtOAc (20 mL) andthe solution was washed with brine then dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) to afford the sub-titlecompound (90 mg, 38%).

¹H NMR (300 MHz, CD₃OD) δ 0.34-2.16 (m, 43H), 2.34 (s, 4H), 2.56 (s,4H), 3.01 (m, 1H), 3.49 (s, 4H), 5.03 (d, J=12.1 Hz, 2H), 5.20 (d,J=12.0 Hz, 2H), 6.65 (d, J=8.4 Hz, 1H), 6.65 (d, J=4.8 Hz, 1H),7.36-7.38 (m, 6H), 7.98 (s, 1H). APCI MS m/z 759 [C₄₈H₆₆N₆O₂+H]⁺.

(ii) Preparation of 56:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 56b (70 mg, 0.09 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (70 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) to afford the titlecompound (42 mg, 69%).

R_(f) 0.25 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.35-2.24 (m, 40H), 2.37 (s, 3H), 2.62 (s,4H), 3.07 (m, 1H), 3.53 (s, 4H), 4.53 (s, 1H), 6.86 (d, J=8.4 Hz, 1H),7.75 (d, J=8.1 Hz, 1H), 8.03 (s, 1H). APCI MS m/z 669 [C₄₁H₆₀N₆O₂+H]⁺.m.p. 280-300° C. dec. HPLC (Method A)>99% (214 nm) t_(R)=10.3 min.

Example 57

(i) Preparation of 57b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(6-(4-(tert-butoxycarbonylamino)piperidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To 41b (200 mg, 0.29 mmol) in MeOH (4 mL) was added tert-butylpiperidin-4-ylcarbamate (1.5 g, 7.50 mmol). The reaction mixture washeated at 140° C. for 7 hours using microwave irradiation and thenconcentrated. The residue was dissolved in EtOAc (20 mL) and thesolution was washed with brine then dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (0-10% MeOH in CH₂Cl₂) to afford the sub-title compound(200 mg, 79%).

APCI MS m/z 859 [C₅₃H₇₄N₆O₄+H]⁺.

(ii) Preparation of 57c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(6-(4-aminopiperidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 57b (190 mg, 0.22 mmol) in MeOH (10 mL) was added HCl(1.0 M in diethyl ether; 10 mL, 10.0 mmol). The mixture was stirred atroom temperature for 32 hours and then concentrated under reducedpressure. Purification of the residue by column chromatography (silica,0-70% CMA in CH₂Cl₂) afforded the sub-title compound (100 mg, 59%).

APCI MS m/z 759 [C₄₈H₆₆N₆O₂+H]⁺.

(iii) Preparation of 57:(4aS,6aS,6bR,13aR)-12-Amino-15-(6-(4-aminopiperidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 57c (100 mg, 0.13 mmol) and MeOH (10 mL) was flushed withnitrogen and then 10% Pd/C (100 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) to afford the titlecompound (25 mg, 29%).

R_(f) 0.11 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.36-2.35 (m, 46H), 3.07 (m, 2H), 3.10 (dd,J=3.4 Hz, 3.9 Hz, 1H), 4.29 (d, J=13.2 Hz, 2H), 6.87 (d, J=8.7 Hz, 1H),7.61 (m, 1H), 8.03 (s, 1H). APCI MS m/z 669 [C₄₁H₆₀N₆O₂+H]⁺. m.p.260-270° C. dec. HPLC (Method A) 95.3% (214 nm) t_(R)=10.8 min.

Example 58

(i) Preparation of 58b: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(6-(3-(tert-butoxycarbonylamino)pyrrolidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 41b (246 mg, 0.36 mmol) in MeOH (4 mL) was addedtert-butyl pyrrolidin-3-ylcarbamate (1.0 g, 5.36 mmol). The reactionmixture was heated at 140° C. for 6 hours using microwave irradiation.The solvent was removed under reduced pressure and the residue wasdissolved in EtOAc (20 mL). The solution was washed with brine thendried (Na₂SO₄), filtered and concentrated. The residue was purified bycolumn chromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford thesub-title compound (306 mg, 100%).

APCI MS m/z 845 [C₅₂H₇₂N₆O₄+H]⁺.

(ii) Preparation of 58c: (4aS,6aS,6bR,13aR)-Benzyl12-amino-15-(6-(3-aminopyrrolidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 58b (300 mg, 0.35 mmol) in MeOH (10 mL) was added HCl(1.0 M in diethyl ether, 7.8 mL, 7.80 mmol). The reaction mixture wasstirred at room temperature for 24 hours and then concentrated. Theresidue was purified by column chromatography (silica, 0-40% CMA inCH₂Cl₂) to afford the sub-title compound (50 mg, 19%).

¹H NMR (300 MHz, CD₃OD) δ 0.36-2.35 (m, 42H), 3.08-3.68 (m, 7H), 5.05(d, J=12.0 Hz, 1H), 5.21 (d, J=12.0 Hz, 1H), 6.32 (d, J=8.7 Hz, 1H),7.30-7.39 (m, 6H), 7.89 (s, 1H).

APCI MS m/z 745 [C₄₇H₆₄N₆O₂+H]⁺.

(iii) Preparation of 58:(4a,6aS,6bR,13aR)-12-Amino-15-(6-(3-aminopyrrolidin-1-yl)pyridin-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A solution of 58c (50 mg, 0.067 mmol) and MeOH (15 mL) was flushed withnitrogen and then 10% Pd/C (50 mg) was added. The mixture was flushedwith nitrogen followed by hydrogen. The mixture was stirred underhydrogen at atmospheric pressure overnight. The catalyst was removed byfiltration through diatomaceous earth and the filtrate was concentrated.The residue was purified by column chromatography (silica, 0-70% CMA inCH₂Cl₂) to afford the title compound (33 mg, 76%).

R_(f) 0.14 (40:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.37-2.36 (m, 44H), 3.10-3.80 (m, 5H), 6.53(d, J=8.7 Hz, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.93 (s, 1H). APCI MS m/z 655[C₄₀H₅₈N₆O₂+H]⁺. m.p.>300° C. HPLC (Method A) 97.4% (214 nm) t_(R)=10.8min.

Example 59

(i) Preparation of 59b: (4aS,6aS,6bR,8aR,13aR,15bS)-benzyl12-amino-15-(2-formylphenyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

A mixture of II (500 mg, 0.75 mmol), 2-formylphenylboronic acid (337 mg,2.24 mmol) Pd(PPh₃)₄ (80 mg, 0.069 mmol) and K₂CO₃ (414 mg, 3.0 mmol) inbenzene (3.5 mL) and EtOH (1.5 mL) was sealed and heated to 120° C. bymicrowave for 1 hour. The reaction mixture was diluted with EtOAc (100mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (540 mg) as a brown solid.

APCI MS (Positive Mode) m/z 688 [C₄₅H₅₇N₃O₃+H]⁺.

(ii) Preparation of 59c: (4aS,6aS,6bR,8aR,13aR,15bS)-Benzyl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylate

To a solution of 59b (140 mg, 0.20 mmol) and morpholine (0.035 mL, 0.40mmol) in CH₂Cl₂ (5 mL) was added sodium triacetoxyborohydride (85 mg,0.40 mmol). The mixture was stirred at room temperature for 12 hours.The reaction mixture was diluted with EtOAc (150 mL). The organic phasewas washed with brine then dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (71 mg, 47%) as abrown solid.

APCI MS (Positive Mode) m/z 759 [C₄₉H₆₆N₄O₃+H]⁺.

(iii) Preparation of 59:(4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)phenyl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid

A mixture of 59c (71 mg, 0.093 mmol) and 10% Pd(OH)₂/C (50 mg) in MeOH(12 mL) and EtOAc (3 mL) was stirred under a hydrogen balloon for 12hours. The reaction mixture was filtered through a pad of diatomaceousearth and washed with CMA (25 mL). The filtrate was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-70% CMA in CH₂Cl₂) to afford the titlecompound (20 mg, 33%) as a mixture of rotamers.

R_(f) 0.80 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.25 (s, 3H), 0.78 (s, 3H), 0.89 (s, 3H), 1.08(s, 3H), 1.12 (s, 3H), 1.28 (s, 6H), 1.35-2.30 (m, 23H), 2.28 (d, J=14.8Hz, 1H), 2.40 (m, 1H), 2.90 (m, 1H), 3.95-4.50 (m, 5H), 7.20-7.62 (m,4H). mp>300° C. dec. APCI MS (Positive Mode) m/z 669 [C₄₂H₆₀N₄O₃+H]⁺.

Example 60

(i) Preparation of 60b:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-3-oxopropanenitrile

To a suspension of 1 (150 mg, 0.30 mmol) and CH₂Cl₂ (7 mL) was addedthionyl chloride (0.22 mL, 3.0 mmol) at room temperature. The mixturewas stirred for 1 hour, after which time the solvent was removed underreduced pressure. The residue was taken up in toluene (5 mL),concentrated and placed under vacuum at room temperature. A solution ofCH₃CN (0.16 mL, 3.0 mmol) and THF (5 mL) was cooled to −78° C. undernitrogen. The solution was treated with n-butyllithium (2.5 M inhexanes, 1.27 mL, 3.2 mmol) while maintaining the internal temperaturebelow −70° C. The previously prepared acid chloride was taken up in THF(2 mL), cooled to −78° C. and slowly added to the lithium salt mixture.The reaction mixture was stirred for 80 minutes and then quenched with 1N HCl (10 mL). The layers were separated and the aqueous layer wasextracted with EtOAc (15 mL) and the combined organic layers were dried(Na₂SO₄) and filtered. The solvent was removed under reduced pressureand the residue was purified by column chromatography (silica, 0-50% CMAin CH₂Cl₂) to provide the sub-title compound (115 mg, 74%) as anoff-white solid.

¹H NMR (300 MHz, CD₃OD) δ 0.80 (s, 3H), 0.91-0.96 (m, 9H), 1.12-1.17 (m,12H), 1.31-1.91 (m, 15H), 2.08-2.12 (m, 3H), 2.39-2.45 (m, 1H),2.71-2.81 (m, 1H), 5.43-5.45 (m, 1H). ESI MS m/z 517 [C₃₃H₄₈N₄O+H]⁺.HPLC 97.6% (area %), t_(R)=16.2 min.

(ii) Preparation of 60:(4aS,6aS,6bR,13aR)-4-a-(5-Amino-1H-pyrazol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-amine

A solution of 60b (100 mg, 0.19 mmol), hydrazine (31 mg, 0.97 mmol) andEtOH (5 mL) was heated at reflux under nitrogen for 16 hours. A secondportion of hydrazine (91 mg, 2.85 mmol) was added and the reactioncontinued for 72 hours at reflux. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,0-50% CMA in CH₂Cl₂) to provide the title compound (43 mg, 43%) as anoff-white solid.

¹H NMR (300 MHz, CD₃OD) δ 0.58 (s, 3H), 0.82 (s, 3H), 0.91-1.35 (m,18H), 1.39-1.91 (m, 15H), 2.13-2.35 (m, 2H), 2.73-2.81 (m, 1H),5.43-5.48 (m, 2H). ESI MS m/z 531 [C₃₃H₅₀N₆+H]⁺. HPLC 97.9% (area %),t_(R)=14.2 min.

Example 61

(i) Preparation of 61:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-N-(1H-tetrazol-5-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of 1H-tetrazol-5-amine hydrate (103 mg, 1.0 mmol) andtriethylamine (0.27 mL, 2.0 mmol) in THF (3 mL) was added III (100 mg,0.20 mmol). The mixture was stirred at room temperature overnight. Thereaction mixture was diluted with EtOAc (60 mL). The organic phase waswashed with brine, dried (MgSO₄), filtered and concentrated to drynessunder reduced pressure. The residue was purified by columnchromatography (silica, 0-80% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (35 mg, 32%) as an off-white solid.

R_(f) 0.40 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.73 (s, 3H), 0.85 (s, 3H), 0.92 (s, 3H), 0.97(2, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.25 (s, 3H), 1.30-1.90 (m, 16H),2.10 (m, 2H), 2.20 (m, 1H), 2.41 (d, J=15.0 Hz, 1H), 3.05 (m, 1H), 5.46(s, 1H). mp>300° C. ESI MS (Positive Mode) m/z 561 [C₃₂H₄₈N₈O+H]⁺.

Example 62

(i) Preparation of 62:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a suspension of 1 (250 mg, 0.51 mmol) and CH₂Cl₂ (5 mL) was addedthionyl chloride (0.4 mL, 5.1 mmol) at room temperature. The mixture wasstirred for 3 hours, after which time the solvent was removed underreduced pressure. The residue was taken up in toluene (5 mL),concentrated and placed under vacuum at room temperature for 1 hour. Theresidue was taken up in THF (5 mL) and concentrated NH₄OH (1.4 mL) wasadded. The mixture was stirred for 1 hour and another portion of NH₄OH(1.4 mL) was added. The mixture was stirred at room temperature overnight before being quenched with saturated NaHCO₃ solution (10 mL). Themixture was extracted with CH₂Cl₂ (3×15 mL), dried (Na₂SO₄) andfiltered. The solvent was removed under reduced pressure and the residuewas purified by column chromatography (silica, 0-3% MeOH in CH₂Cl₂) toprovide the title compound (144 mg, 58%) as an off-white solid.

¹H NMR (500 MHz, CD₃OD) δ 0.89-0.98 (m, 12H), 1.02-1.31 (m, 12H),1.39-1.91 (m, 12H), 2.03-2.11 (m, 3H), 2.42-2.45 (m, 1H), 2.76-2.83 (m,1H), 5.43-5.45 (m, 1H).

ESI MS m/z 493 [C₃₁H₄₈N₄O+H]⁺. HPLC 97.9% (area %), t_(R)=14.2 min.

Example 63

(i) Preparation of 63:(4aS,6aS,6bR,13aR)-4-a-(Aminomethyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-amine

To a solution of 62 (300 mg, 0.61 mmol) and THF (20 mL) was addedlithium aluminum hydride (230 mg, 6.1 mmol) at room temperature. Themixture was heated at reflux for 24 hours, cooled to room temperature,carefully quenched with H₂O and EtOAc then extracted with CH₂Cl₂ (3×20mL). The organics were dried (Na₂SO₄) and filtered. The solvent wasremoved under reduced pressure and the residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) to provide the titlecompound (94 mg, 30%).

¹H NMR (300 MHz, CDCl₃) δ 0.82-1.03 (m, 12H), 1.15-1.85 (m, 14H),1.91-2.11 (m, 15H), 2.52-2.60 (m, 2H), 2.83-2.91 (m, 1H), 3.25-3.30 (m,1H), 3.56-3.63 (m, 1H), 5.23-5.39 (m, 2H), 7.92-7.94 (m, 2H). ESI MS m/z479 [C₃₁H₅₀N₄+H]⁺. HPLC 96.6% (area %), t_(R)=11.3 min.

Example 64

(i) Preparation of 64b:(4aS,6aS,6bR,13aR)-12-Amino-N,N,2,2,6a,6b,9,9,13a-nonamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a suspension of 1 (200 mg, 0.41 mmol) and CH₂Cl₂ (4 mL) was addedthionyl chloride (0.3 mL, 4.1 mmol) at room temperature. The mixture wasstirred for 0.5 hours, after which time the solvent was removed underreduced pressure. The residue was taken up in toluene (5 mL),concentrated and placed under vacuum at room temperature for 1 hour. Theresidue was taken up in THF (4 mL) and N,N-diisopropylethylamine (0.23mL, 1.2 mmol) was added followed by diethylamine solution (2.0 M in THF,2.05 mL, 4.1 mmol). The solution was stirred overnight and then quenchedwith a 2% solution of citric acid. The layers were separated and theaqueous solution was extracted with CH₂Cl₂ (2×10 mL). The combinedextracts were dried (Na₂SO₄) and filtered. The solvent was removed underreduced pressure and the residue was purified by column chromatography(silica, 0-7% MeOH in CH₂Cl₂) to provide the sub-title compound (153 mg,73%) as an off-white solid.

¹H NMR (300 MHz, CD₃OD) δ 0.83 (s, 3H), 0.91-0.98 (m, 9H), 1.17-1.26 (m,13H), 1.39-2.25 (m, 16H), 3.01-3.20 (m, 7H), 5.29 (m, 1H). ESI MS m/z521 [C₃₃H₅₂N₄O+H]⁺. HPLC 98.4% (area %), t_(R)=17.2 min.

(ii) Preparation of 64:(4aS,6aS,6bR,13aR)-4-a-((Dimethylamino)methyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-amine

A solution of 64b (129 mg, 0.25 mmol) and THF (12.5 mL) was slowly addedto a suspension of lithium aluminum hydride (94 mg, 2.5 mmol) and THF(6.5 mL) at room temperature. The mixture was stirred overnight and thenquenched with EtOAc followed by H₂O. The layers were separated and theaqueous layer was extracted with EtOAc (3×15 mL). The organics werecombined, dried (Na₂SO₄) and filtered. The solvent was removed underreduced pressure and the residue was purified by column chromatography(silica, 0-7% MeOH in CH₂Cl₂) to provide the title compound (54 mg, 43%)as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.83-0.95 (m, 12H), 1.02-1.33 (m, 20H),1.35-2.10 (m, 16H), 2.30-2.41 (m, 3H), 5.21-5.25 (m, 1H). ESI MS m/z 507[C₃₃H₅₄N₄+H]⁺. HPLC 96.4% (area %), t_(R)=11.6 min

Example 65

(i) Preparation of 65:(4aS,6aS,6bR,13aR)-12-Amino-N-hydroxy-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a suspension of 1 (75 mg, 0.15 mmol) in CH₂Cl₂ (5 mL) was addedthionyl chloride (0.1 mL, 1.5 mmol) at room temperature. The mixture wasstirred for 0.5 hours, after which time the solvent was removed underreduced pressure. The residue was taken up in toluene (5 mL),concentrated and placed under vacuum at room temperature overnight. Theresidue was taken up in CH₂Cl₂ (5 mL) at room temperature.N,N-Diisopropylethylamine (0.2 mL, 1.05 mmol) was added followed byhydroxylamine hydrochloride (52 mg, 0.75 mmol). The mixture was stirredat room temperature for 14 hours. A solution of citric acid (2%, 5 mL)was added and the mixture was extracted with CH₂Cl₂ (2×5 mL). Thecombined organic layers were dried (Na₂SO₄), filtered and concentratedunder reduced pressure. The residue was purified by columnchromatography (silica, 0-6% MeOH in CH₂Cl₂) to provided the titlecompound (144 mg, 58%) as an off-white solid.

¹H NMR (500 MHz, CD₃OD) δ 0.87-0.95 (m, 12H), 1.05-1.33 (m, 12H),1.38-1.88 (m, 13H), 2.08-2.15 (m, 3H), 2.43-2.46 (m, 1H), 2.75-2.82 (m,1H), 5.43-5.45 (m, 1H). APCI MS m/z 509 [C₃₁H₄₈N₄O+H]⁺. HPLC 97.4% (area%), t_(R)=13.5 min.

Example 66

(i) Preparation of 66:(4aS,6aS,6bR,13aR)-12-Amino-N-(5-hydroxy-1H-pyrazol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of III (250 mg, 0.48 mmol) and THF (2 mL) was addedtriethylamine (0.68 mL, 4.88 mmol) and the solution was stirred for 5min. To the resultant mixture was added a solution of3-amino-5-hydroxypyrazole (241 mg, 2.44 mmol) in DMF (6 mL) and themixture was heated at 50° C. for 3 hours. The mixture was poured intoH₂O (20 mL) and the precipitate was removed by filtration. The filtratewas concentrated and the residue was purified by column chromatography(silica, 0-50% CMA in CH₂Cl₂) followed by preparative HPLC to afford thetitle compound (48 mg, 17%) as a solid.

R_(f) 0.28 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CDCl₃) δ 0.49-2.97 (m, 42H), 5.36 (s, 1H), 5.76 (s,1H). APCI MS m/z 575 [C₃₄H₅₀N₆O₂+H]⁺. m.p. 275-295° C. dec. HPLC (MethodA) 97.7% (214 nm) t_(R)=11.5 min.

Example 67

(i) Preparation of 67b:(4aS,6aS,6bR,12aR)-11-Cyano-2,2,6a,6b,9,9,12a-heptamethyl-10-oxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydropicene-4-a-carboxylicacid

A solution of diisopropylamine (1.4 mL, 10.2 mmol) and THF (20 mL) wascooled to −78° C. under nitrogen. A solution of n-butyllithium (2.5 M inhexanes, 4.4 mL, 11.0 mmol) was slowly added, maintaining the internaltemperature below −70° C. The solution was allowed to stir for 30 minand was then slowly added to a solution of Ib (2.0 g, 4.4 mmol) and THF(250 mL) at −78° C. under nitrogen. This solution was stirred for 30 minafter which time a suspension of p-toluenesulfonyl cyanide (1.6 g, 8.8mmol) and THF (25 mL) was added over 45 min. The solution was stirredfor 1 hour and then quenched by the addition of saturated ammoniumchloride solution (100 mL) at −78° C. The mixture was allowed to warm toroom temperature overnight. The organic layer was separated and theaqueous layer was extracted with EtOAc (3×100 mL). The combined organiclayers were dried (Na₂SO₄) and filtered. The solvent was removed underreduced pressure and the residue was purified by column chromatography(silica, 0-35% EtOAc in hexanes) to provide the sub-title compound (1.3g, 60%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 0.77-0.82 (m, 9H), 1.06-1.11 (m, 13H),1.12-2.03 (m, 19H), 2.09-2.38 (m, 1H), 2.81-2.89 (m, 1H), 5.26-5.35 (m,1H).

(ii) Preparation of 67c:(4aS,6aS,6bR,13aR)-12-Amino-11-benzyl-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid (and N-benzyl structural isomer)

A solution of 67b (1.3 g, 2.6 mmol), benzylhydrazine dihydrochloride(1.2 g, 6.2 mmol) and EtOH (15 mL) was heated at reflux for 58 hours.The solvent was removed under reduced pressure and the residue waspurified by column chromatography (silica, 0-5% MeOH in CH₂Cl₂) toprovide the sub-title compound (1.1 g, 72%) as yellow solid.

¹H NMR (300 MHz, CDCl₃) δ 0.78-0.95 (m, 15H), 1.05-2.03 (m, 25H),2.25-2.35 (m, 1H), 2.81-2.91 (m, 1H), 5.21-5.36 (m, 3H), 7.04-7.11 (m,2H), 7.21-7.35 (m, 3H).

ESI MS m/z 584 [C₃₈H₅₃N₃O₂+H]⁺.

(iii) Preparation of 67d:(4aS,6aS,6bR,13aR)-1′-Benzyl-12-(benzyloxycarbonylamino)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxylicacid (and N-benzyl structural isomer)

To a solution of 67c (560 mg, 0.96 mmol) in THF (50 mL) were addedtriethylamine (0.27 mL, 1.92 mmol) and benzyl chloroformate (0.16 mL,1.15 mmol) under nitrogen.

The mixture was stirred at room temperature for 16 hours and thenquenched with brine (40 mL). The layers were separated and the aqueouslayer was extracted with EtOAc (2×20 mL). The solution was dried(Na₂SO₄), filtered and concentrated to afford the sub-title compound(108 mg, quant) which was used without further purification.

ESI MS m/z 718 [C₄₆H₅₉N₃O₄+H]⁺.

(iv) Preparation of 67e: Benzyl(4aS,6aS,6bR,13aR)-11-benzyl-4-a-(hydrazinecarbonyl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-ylcarbamate(and N-benzyl structural isomer)

To a solution of 67d (744 mg, 1.03 mmol) in CH₂Cl₂ (100 mL) was addedthionyl chloride (0.76 mL, 10.37 mmol). The mixture was stirred at roomtemperature for 1.5 hours and then the solvent was removed under reducedpressure. The residue was azeotroped with toluene and then dried undervacuum overnight to afford the intermediate acid chloride (687 mg, 93%).The crude acid chloride (364 mg, 0.49 mmol) was dissolved in THF (10 mL)and then triethylamine (0.34 mL, 2.47 mmol) and hydrazine (0.15 mL, 4.94mmol) were added. The mixture was stirred at room temperature for 20hours and concentrated under reduced pressure. The residue waspartitioned between EtOAc (20 mL) and H₂O (10 mL). The layers wereseparated and the organic layer was washed with H₂O and brine. Thesolution was dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (silica, 0-40% CMA in CH₂Cl₂) toafford the sub-title compound (224 mg, 62%).

ESI MS (m/z 733 [C₄₆H₆₁N₅O₃+H]⁺.

(v) Preparation of 67f: Benzyl(4aS,6aS,6bR,13aR)-11-benzyl-2,2,6a,6b,9,9,13a-heptamethyl-4-a-(1,3,4-oxadiazol-2-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-ylcarbamate(and N-benzyl structural isomer)

A mixture of 67e (100 mg, 0.13 mmol), trimethoxymethane (0.22 mL, 2.04mmol) and p-toluene sulfonic acid monohydrate (4 mg, 0.02 mmol) washeated at 120° C. overnight. The mixture was concentrated under reducedpressure and the residue was purified by column chromatography (silica,2:1 hexanes/EtOAc) to afford the sub-title compound (75 mg, 74%).

ESI MS m/z 651 [C₄₇H₅₉N₅O₃—C₇H₇+H]⁺.

(vi) Preparation of 67:(4aS,6aS,6bR,13aR)-2,2,6a,6b,9,9,13a-Heptamethyl-4-a-(1,3,4-oxadiazol-2-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-amine

A solution of 67d (70 mg, 0.09 mmol), acetic acid (0.2 mL) and MeOH (10mL) was flushed with nitrogen. To the resultant mixture was added 10%Pd/C (150 mg) and the mixture was flushed with nitrogen followed byhydrogen. The mixture was stirred under hydrogen at atmospheric pressurefor 23 hours. The catalyst was removed by filtration throughdiatomaceous earth and the filtrate was concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-40% CMA in CH₂Cl₂) to afford the title compound (6 mg, 12%).

R_(f) 0.42 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.45-2.41 (m, 41H), 3.14 (d, J=10.8 Hz, 1H),5.44 (s, 1H), 8.84 (s, 1H). ESI MS m/z 518 [C₃₂H₄₇N₅O+H]⁺. m.p. 255-270°C. dec. HPLC (Method A) 89.0% (214 nm) t_(R)=16.1 min.

Example 68

(i) Preparation of 68b: Ethyl3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-3-oxopropanimidatehydrochloride

A solution of 60b (200 mg, 0.39 mmol) and EtOH (15 mL) was saturatedwith anhydrous HCl at −5° C. The solution was kept at that temperaturefor 10 d before removal of the solvent and excess HCl under reducedpressure. The residue was coevaporated with EtOH followed by diethylether to provide the sub-title compound (220 mg, 100%) as a tan solid.

APCI MS m/z 563 [C₃₃H₅₄N₄O₂+H]⁺.

(ii) Preparation of 68c: Ethyl3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-3-oxopropanoatehydrochloride

To a solution of 68b (220 mg, 0.39 mmol) and EtOH (3 mL) was added 1 NHCl (2 mL) at room temperature. After 24 hours, the solvent was removedunder reduced pressure and the residue was purified by columnchromatography (silica, 0-65% CMA in CH₂Cl₂) to provide the sub-titlecompound (135 mg, 58%).

¹H NMR (500 MHz, CDCl₃) δ 0.71-0.95 (m, 12H), 1.13-1.31 (m, 16H),1.34-2.15 (m, 17H), 2.29-2.33 (m, 1H), 2.71-2.74 (m, 1H), 3.49-3.71 (m,2H), 4.13-4.21 (m, 2H), 5.43-5.45 (m, 1H).

(iii) Preparation of 68:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-1H-pyrazol-5-ol

A solution of 68c (135 mg, 0.24 mmol), hydrazine (38 mg, 1.2 mmol) andEtOH (5 mL) was heated at reflux under nitrogen for 66 hours and thencooled to room temperature. The solvent was removed under reducedpressure and the residue was purified by column chromatography (silica,0-55% CMA in CH₂Cl₂) to provide the title compound (11 mg, 9%) as asolid.

¹H NMR (300 MHz, CD₃OD) δ 0.62 (s, 3H), 0.86-1.01 (m, 9H), 1.03-1.39 (m,15H), 1.41-1.99 (m, 11H), 2.03-2.11 (m, 2H), 2.25-2.45 (m, 2H),2.74-2.81 (m, 1H), 5.43-5.45 (m, 1H). APCI MS m/z 532 [C₃₃H₄₉N₅O+H]⁺.HPLC 98.6% (area %), t_(R)=10.2 min.

Example 69

(i) Preparation of 69:(4aS,6aS,6bR,13aR)-12-amino-N-(3-amino-1H-pyrazol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of 1H-pyrazole-3,5-diamine (70 mg, 0.54 mmol) prepared bythe procedure described in the literature (US82902, 2007) and pyridine(2.0 mL) was added III (100 mg, 0.20 mmol). The mixture was stirred atroom temperature overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (25 mg, 25%) as an off-white solid.

R_(f) 0.20 (180:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, DMSO-d₆) δ 0.83 (s, 3H), 0.92 (s, 3H), 0.95 (s, 3H),1.0 (s, 3H), 1.25 (s, 6H), 1.30 (s, 3H), 1.30-1.90 (m, 23H), 2.06 (m,3H), 2.25 (m, 1H), 2.44 (d, J=15.0 Hz, 1H), 2.62 (m, 1H), 5.35 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 574 [C₃₄H₅₁N₇O+H]⁺.

Example 70

(i) Preparation of 70:(4aS,6aS,6bR,13aR)—S-5-Amino-4H-1,2,4-triazol-3-yl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbothioate

To a solution of 5-amino-4H-1,2,4-triazole-3-thiol (1.42 g, 12.2 mmol)in pyridine (30 mL was added III (2.0 g, 3.91 mmol). The mixture wasstirred at room temperature for 3 hours. The reaction mixture wasconcentrated to remove pyridine under reduced pressure. The residue waspurified by column chromatography (silica, 0-15% MeOH in CH₂Cl₂) toafford the title compound (1.41 g, 59%) as an off-white solid.

R_(f) 0.12 (89:10:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.87 (s, 6H), 0.90 (s, 3H), 0.95 (s, 3H), 1.10(s, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.30-2.20 (m, 19H), 2.38 (d, J=15.0Hz, 1H), 2.88 (m, 1H), 5.39 (s, 1H). mp 278-296° C. ESI MS (PositiveMode) m/z 59 [C₃₃H₄₉N₇OS—H]⁻.

Example 71

(i) Preparation of 71:(4aS,6aS,6bR,13aR)-12-Amino-N-(5-amino-1,3,4-thiadiazol-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of III (500 mg, 0.97 mmol) and pyridine (4 mL) was added1,3,4-thiadiazole-2,5-diamine (340 mg, 2.92 mmol). The reaction mixturewas heated at 60° C. for 20 hours and then poured into H₂O (50 mL). Theprecipitate was collected by filtration and dissolved in CH₂Cl₂/i-PrOH(2:1, 20 mL). The aqueous layer was extracted with CH₂Cl₂/i-PrOH (2:1,2×25 mL). The combined extracts were dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (32 mg, 6%) as a solid.

R_(f) 0.20 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide)

¹H NMR (300 MHz, DMSO-d₆) δ 0.77-2.43 (m, 41H), 2.94 (d, J=12 Hz, 1H),5.32 (s, 1H), 7.46 (s, 1H), 11.50 (s, 1H), 13.20 (s, 1H).

APCI MS m/z 592 [C₃₃H₄₉N₇OS+H]⁺. m.p. 280-300° C. dec. HPLC (Method A)95.4% (214 nm) t_(R)=10.2 min.

Example 72

(i) Preparation of 72:(4aS,6aS,6bR,8aR,13aR,15bS)-4-a-(5-Amino-1-methyl-1H-pyrazol-3-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-12-amine

A mixture of 60b (282 mg, 0.54 mmol) and methylhydrazine (0.14 mL, 2.73mmol) in EtOH (4 mL) was sealed and heated to 160° C. by microwave for 3hours. The reaction mixture was concentrated to dryness. The residue waspurified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂)followed by preparative HPLC to afford title compound (35 mg, 12%) as awhite solid.

R_(f) 0.34 (89:10:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.65 (s, 3H), 0.88 (s, 3H), 0.95 (s, 3H), 0.97(s, 3H), 1.15 (s, 3H), 1.20 (s, 3H), 1.24 (s, 3H), 1.25-2.10 (m, 19H),2.38 (m, 2H), 2.78 (m, 1H), 3.68 (s, 3H), 5.47 (m, 1H). mp 195-210° C.dec. APCI MS (Positive Mode) m/z 545 [C₃₄H₅₂N₆+H]⁺.

Example 73

(i) Preparation of 73:(2-Amino-1H-imidazol-1-yl)((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)methanone

To a solution of III (200 mg, 0.39 mmol) and pyridine (2 mL) was added1H-imidazol-2-amine (70 mg, 0.84 mmol). The reaction mixture was stirredat room temperature for 3.5 hours and then poured into H₂O (20 mL). Theprecipitate was collected by filtration and dissolved in CH₂Cl₂/i-PrOH(2:1, 10 mL). The aqueous layer was extracted with CH₂Cl₂/i-PrOH (2:1,2×10 mL). The combined organics were dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) to afford the titlecompound (96 mg, 44%).

R_(f) 0.50 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, DMSO-d₆) δ 0.91-2.49 (m, 41H), 3.00 (d, J=11.1 Hz, 1H),4.12 (s, 2H), 5.29 (s, 1H), 6.46 (s, 1H), 6.64 (s, 2H), 7.39 (s, 1H),10.95 (s, 1H). APCI MS m/z 559 [C₃₄H₅₀N₆O+H]⁺. m.p. 230-250° C. dec.HPLC (Method A) 97.6% (214 nm) t_(R)=9.9 min.

Example 74

(i) Preparation of 74:(4aS,6aS,6bR,13aR)-12-Amino-N-(5-mercapto-1,3,4-thiadiazol-2-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of 5-amino-1,3,4-thiadiazole-2-thiol (200 mg, 2.1 mmol)prepared by the procedure described in the literature (US3940409, 1976)and pyridine (10.0 mL) was added III (215 mg, 0.42 mmol). The mixturewas stirred at room temperature overnight. The reaction mixture wasconcentrated to dryness under reduced pressure. The residue was purifiedby column chromatography (silica, 0-70% CMA in CH₂Cl₂) followed bypreparative HPLC to afford the title compound (30 mg, 12%) as anoff-white solid.

R_(f) 0.60 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.89 (s, 3H), 0.90 (s, 3H), 0.92 (s, 3H), 0.97(s, 3H), 1.29 (s, 6H), 1.36 (s, 3H), 1.43-2.30 (m, 19H), 2.43 (d, J=14.7Hz, 1H), 2.84 (m, 1H), 5.42 (s, 1H). mp>300° C. ESI MS (Positive Mode)m/z 609 [C₃₃H₄₈N₆OS₂+H]⁺.

Example 75

(i) Preparation of 75b:2-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonyl)hydrazinecarbothioamide

To a solution of hydrazinecarbothioamide (914 mg, 10.0 mmol) andpyridine (50.0 mL) was added III (1.0 g, 2.0 mmol). The mixture wasstirred at room temperature for 2 hours. The reaction mixture was pouredinto H₂O. Brown precipitates were collected by filtration and washedwith H₂O, dried in an oven at 40° C. to provide the sub-title compound(900 mg). The crude compound was used without further purification.

ESI MS (Positive Mode) m/z 567 [C₃₂H₅₀N₆OS+H]⁺.

(ii) Preparation of 75:5-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4H-1,2,4-triazole-3-thiol

A mixture of 75b (100 mg) and NaOH (2 M, 2.0 mL) was heated at refluxfor 7 d. The reaction mixture was concentrated to dryness under reducedpressure. The residue was triturated with MeOH and CH₂Cl₂. The filtratewas concentrated and purified by preparative HPLC to provide the titlecompound (61 mg, 66%) as an off-white solid.

R_(f) 0.60 (180:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.60 (s, 3H), 0.82 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.21 (s, 3H), 1.25 (s, 3H), 1.46 (s, 3H), 1.50-2.05 (m, 18H),2.25 (m, 1H), 2.40 (d, J=15.0 Hz, 1H), 2.98 (m, 1H), 5.40 (s, 1H).mp>300° C. ESI MS (Positive Mode) m/z 549 [C₃₂H₄₈N₆S+H]⁺.

Example 76

(i) Preparation of 76:((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)(3-mercapto-1H-1,2,4-triazol-1-yl)methanone

To a solution of III (300 mg, 0.58 mmol) and pyridine (3 mL) was added1H-1,2,4-triazole-3-thiol (177 mg, 1.75 mmol). The reaction mixture wasstirred at room temperature for 1 hour and then poured into H₂O (30 mL).The precipitate was dissolved in CH₂Cl₂/i-PrOH (2:1, 20 mL). The aqueouslayer was extracted with CH₂Cl₂/i-PrOH (2:1, 3×10 mL) and the combinedorganics were dried (Na₂SO₄), filtered and concentrated under reducedpressure. The residue was purified by column chromatography (silica,0-8% MeOH in CH₂Cl₂) to afford the title compound (110 mg, 33%).

R_(f) 0.27 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide)

¹H NMR (300 MHz, CD₃OD) δ 0.89-2.23 (m, 40H), 2.41 (d, J=14.7 Hz, 1H),3.00 (dd, J=3.6, 12.9 Hz, 1H), 5.40 (s, 1H), 8.46 (s, 1H). APCI MS m/z577 [C₃₃H₄₈N₆OS+H]⁺. m.p. 260-280° C. dec. HPLC (Method A) 96.2% (214nm) t_(R)=14.3 min.

Example 77

(i) Preparation of 77:(4aS,6aS,6bR,13aR)-12-Amino-N-(3-mercapto-5-methyl-4H-1,2,4-triazol-4-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of III (300 mg, 0.58 mmol) and pyridine (3 mL) was added4-amino-5-methyl-4H-1,2,4-triazole-3-thiol (228 mg, 1.75 mmol). Thereaction mixture was stirred at room temperature for 1 hour and thenpoured into H₂O (30 mL). The precipitate was collected by filtration anddried under vacuum overnight. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (30 mg, 8%) as a solid.

R_(f) 0.25 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide)

¹H NMR (300 MHz, CD₃OD) δ 0.89-2.50 (m, 44H), 2.93-2.97 (m, 1H), 5.36(s, 1H). APCI MS m/z 606 [C₃₄H₅₁N₇OS+H]⁺. m.p. 250-270° C. dec. HPLC(Method A) 92.7% (214 nm) t_(R)=13.2 min.

Example 78

(i) Preparation of 78b:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbohydrazide

To a solution of hydrazine (0.15 mL, 4.5 mmol) and triethylamine (0.77mL) was added III (500 mg, 0.91 mmol). The mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with EtOAc (100mL). The organic phase was washed with brine then dried (MgSO₄),filtered and concentrated to dryness. The residue was purified by columnchromatography on (silica, 0-50% CMA in CH₂Cl₂) to afford the sub-titlecompound (360 mg, 78%) as a brown solid.

(ii) Preparation of 78:5-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-1,3,4-thiadiazol-2-ol

To a solution of 78b (140 mg, 0.27 mmol) in CHCl₃ (5 mL) was addedcarbon disulfide (0.58 mL, 0.96 mmol). The mixture was stirred at roomtemperature for 4 hours. The reaction mixture was concentrated todryness under reduced pressure. The residue was dissolved in EtOH (5 mL)and NaOH (2 M, 5 mL) was added. The mixture was heated at reflux for 12hours. The reaction mixture was concentrated to remove EtOH. Brownsolids were collected by filtration and washed with H₂O. The residue waspurified by column chromatography (silica, 0-50% CMA in CH₂Cl₂) followedby preparative HPLC to afford the title compound (30 mg, 12%) as anoff-white solid.

R_(f) 0.30 (10:1 Methylene Chloride/Methanol).

¹H NMR (300 MHz, DMSO-d₆) δ 0.58 (s, 3H), 0.75 (s, 3H), 0.92 (s, 6H),1.16 (s, 3H), 1.21 (s, 3H), 1.26 (s, 3H), 1.31-1.97 (m, 22H), 2.22 (m,1H), 2.43 (d, J=15.0 Hz, 1H), 2.83 (m, 1H), 5.35 (s, 1H). mp>300° C. ESIMS (Positive Mode) m/z 550 [C₃₂H₄₇N₅OS+H]⁺.

Example 79

(i) Preparation of 79:(4aS,6aS,6bR,13aR)-12-Amino-N-(3-mercapto-1H-pyrazol-5-yl)-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of 5-amino-1H-pyrazole-3-thiol (459 mg, 4.0 mmol) preparedby the procedure described in the literature (J. Med. Chem. 2008,51(15), 4672-4684) and pyridine (10.0 mL) was added III (490 mg, 0.95mmol). The mixture was stirred at room temperature overnight. Thereaction mixture was concentrated to dryness under reduced pressure. Theresidue was purified by column chromatography (silica, 0-70% CMA inCH₂Cl₂) followed by preparative HPLC to afford the title compound (115mg, 17%) as an off-white solid.

R_(f) 0.75 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.89 (s, 6H), 0.91 (s, 3H), 0.95 (s, 3H), 1.28(s, 3H), 1.40 (s, 3H), 1.48 (s, 3H), 1.55-2.20 (m, 19H), 2.43 (d, J=14.4Hz, 1H), 2.93 (m, 1H), 5.39 (m, 1H), 5.57 (m, 1H). mp>300° C. ESI MS(Positive Mode) m/z 591 [C₃₄H₅₀N₆OS+H]⁺.

Example 80

(i) Preparation of 80b: 5-Amino-1,3,4-thiadiazole-2-sulfonamide

To a solution of N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide (1.0 g,4.50 mmol) and MeOH (15 mL) was added HCl (2 N, 10 mL). The reactionmixture was heated at reflux for 18 hours and then concentrated underreduced pressure. The residue was purified by column chromatography(silica, 0-10% MeOH in CMA) to afford the sub-title compound (800 mg,98%).

(ii) Preparation of 80:(4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carboxamide

To a solution of III (400 mg, 0.78 mmol), pyridine (15 mL) and DMF (1mL) was added 80b (281 mg, 1.56 mmol). The reaction mixture was stirredat room temperature for 24 hours and then poured into H₂O (15 mL). Theprecipitate was collected by filtration and dried under vacuumovernight. The residue was purified by column chromatography (silica,0-60% CMA in CH₂Cl₂) followed by preparative HPLC to afford the title (8mg, 2%) as a solid.

R_(f) 0.35 (90:9:1 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, Acetone-d₆) δ 0.68-2.27 (m, 40H), 2.45 (d, J=15 Hz,1H), 3.05-3.08 (m, 1H), 5.47 (s, 1H). APCI MS m/z 656 [C₃₃H₄₉N₇O₃S₂+H]⁺.m.p. 280-300° C. dec.

HPLC (Method A)>99% (214 nm) t_(R)=14.0 min.

Example 81

(i) Preparation of 81b:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonyl)dihydrofuran-2(3H)-one

To a solution of lithium bis(trimethylsilyl)amide (2.3 mL, 1 M in THF,2.3 mmol) was added dihydrofuran-2(3H)-one (0.15 mL, 1.9 mmol) at −78°C. The mixture was stirred for 15 min. The anion solution was added to asolution of III (100 mg, 0.19 mmol) in THF (7 mL) precooled to −78° C.The mixture was stirred at −78° C. for 30 min and deemed incomplete. Toa second solution of lithium bis(trimethylsilyl)amide (1.1 mL, 1 M inTHF, 1.1 mmol) was added dihydrofuran-2(3H)-one (0.075 mL, 1.0 mmol) at−78° C. The second anion mixture was stirred for 15 min. The anionsolution was added to the above reaction mixture at −78° C. The mixturewas continued to stir at −78° C. for 30 min. The reaction mixture wasquenched with saturated NH₄Cl (60 mL) and extracted with EtOAc (100 mL).The organic phase was washed with brine, dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) to afford the sub-titlecompound (150 mg) as a brown solid.

ESI MS (Positive Mode) m/z 562 [C₃₅H₅₁N₃O₃+H]⁺.

(ii) Preparation of 81:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-hydroxyethyl)-1H-pyrazol-5-ol

A mixture of 81b (150 mg) and hydrazine (0.037 mL) in EtOH (2 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-60% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (12 mg, 8%) as an off-white solid.

R_(f) 0.70 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.32 (s, 3H), 1.35-2.08 (m, 18H),2.25 (m, 1H), 2.36 (d, J=15.0 Hz, 1H), 2.74 (m, 2H), 2.82 (m, 1H), 3.72(t, J=6.6 Hz, 2H), 5.51 (s, 1H). mp 251-255° C. APCI MS (Positive Mode)m/z 576 [C₃₅H₅₃N₅O₂+H]⁺.

Example 82

(i) Preparation of 82b: Ethyl3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-2-methyl-3-oxopropanoate

To a solution of lithium bis(trimethylsilyl)amide (3.8 mL, 1 M in THF,3.8 mmol) was added ethyl propionate (0.22 mL, 1.9 mmol). The mixturewas stirred for 30 min. The anion solution was added to a solution ofIII (100 mg, 0.19 mmol) in THF (3 mL) precooled to −78° C. The mixturewas stirred at −78° C. for 1 hour and the reaction was deemedincomplete. To a second solution of lithium bis(trimethylsilyl)amide(1.9 mL, 1 M in THF, 1.9 mmol) was added ethyl propionate (0.33 mL, 1.9mmol). The second anion mixture was stirred for 30 min. The anionsolution was added to the above reaction mixture at −78° C. The mixturewas continued to stir at −78° C. for 30 min. The reaction mixture wasquenched with saturated NH₄Cl (20 mL) and extracted with EtOAc (100 mL).The organic phase was washed brine, dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) to afford the sub-titlecompound (100 mg) as a brown solid.

APCI MS (Positive Mode) m/z 578 [C₃₆H₅₅N₃O₃+H]⁺.

(ii) Preparation of 82:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-methyl-1H-pyrazol-5-ol

A mixture of 82b (100 mg) and hydrazine (0.06 mL) in EtOH (2 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) followed by preparativeHPLC to the title compound (7 mg, 7%) as an off-white solid.

R_(f) 0.70 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.55 (s, 3H), 0.85 (s, 3H), 0.96 (s, 3H), 1.00(s, 3H), 1.19 (s, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.32-2.08 (m, 21H),2.39 (m, 1H), 2.44 (d, J=15.0 Hz, 1H), 2.90 (m, 1H), 5.48 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 546 [C₃₄H₅₁N₅O+H]⁺.

Example 83

(i) Preparation of 83b: Ethyl3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-2-fluoro-3-oxopropanoate

To a solution of lithium bis(trimethylsilyl)amide (5.9 mL, 1 M in THF,5.9 mmol) was added ethyl 2-bromo-2-fluoroacetate (0.46 mL, 3.9 mmol) at−78° C. The mixture was stirred for 10 min. The anion solution was addedto a solution of III (200 mg, 0.39 mmol) in THF (4 mL) precooled to −78°C. The mixture was stirred at −78° C. for 1 hour and the reaction wasdeemed incomplete. To a second solution of n-butyllithium (1.8 mL, 2.5 Min hexanes, 5.9 mmol) was added ethyl 2-bromo-2-fluoroacetate (0.46 mL,3.9 mmol) at −78° C. The second anion mixture was stirred for 10 min.The anion solution was added to the above reaction mixture at −78° C.The mixture was stirred at −78° C. for 1 hour and warmed to −20° C. over1 hour. The reaction mixture was quenched with saturated NH₄Cl (20 mL)and extracted with EtOAc (100 mL). The organic phase was washed withbrine, dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-30% CMA in CH₂Cl₂) toafford the sub-title compound (110 mg) as a brown solid.

APCI MS (Positive Mode) m/z 582 [C₃₅H₅₂FN₃O₃+H]⁺.

(ii) Preparation of 83:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-fluoro-1H-pyrazol-5-ol

A mixture of 83b (110 mg) and hydrazine (0.05 mL) in EtOH (2 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-50% CMA in CH₂Cl₂) followed by preparativeHPLC to afford the title compound (9 mg, 10%) as an off-white solid.

R_(f) 0.50 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.52 (s, 3H), 0.85 (s, 3H), 0.96 (s, 3H), 1.00(s, 3H), 1.19 (s, 3H), 1.23 (s, 3H), 1.28 (s, 3H), 1.32-2.08 (m, 18H),2.39 (m, 1H), 2.40 (d, J=15.0 Hz, 1H), 2.90 (m, 1H), 5.42 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 550 [C₃₃H₄₈FN₅O+H]⁺.

Example 84

(i) Preparation of 84b:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonyl)-1-vinylpyrrolidin-2-one

To a solution of lithium bis(trimethylsilyl)amide (38 mL, 1 M in THF,38.0 mmol) was added 1-vinyl-2-pyrrolidinone (2.1 mL, 19.0 mmol) at −78°C. The mixture was stirred for 10 min. The anion solution was added to asolution of III (1.0 g, 1.9 mmol) in THF (20 mL) precooled to −78° C.The mixture was stirred at −78° C. for 1 hour. The reaction mixture wasquenched with saturated NH₄Cl (60 mL) and extracted with EtOAc (100 mL).The organic phase was washed with brine, dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-10% MeOH in CH₂Cl₂) to afford the sub-titlecompound (350 mg) as a brown solid.

APCI MS (Positive Mode) m/z 587 [C₃₇H₅₄N₄O₂+H]⁺.

(ii) Preparation of 84:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-aminoethyl)-1H-pyrazol-5-ol

A mixture of 84b (100 mg) and hydrazine (0.05 mL) in EtOH (2 mL) wassealed and heated to 160° C. by microwave for 3 hours. The reactionmixture was concentrated to dryness under reduced pressure. The residuewas purified by column chromatography (silica, 0-80% CMA in CH₂Cl₂) toafford the title compound (50 mg, 30%) as a brown solid.

R_(f) 0.27 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.09 (s, 3H), 1.21 (s, 6H), 1.35-2.40 (m, 21H), 2.72 (m, 2H),2.96 (m, 2H), 5.47 (s, 1H). mp 250-260° C. dec. APCI MS (Positive Mode)m/z 575 [C₃₅H₅₄N₆O+H]⁺.

Example 85 and Example 86

(i) Preparation of 85 and 86:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-(dimethylamino)ethyl)-1H-pyrazol-5-oland3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-(methylamino)ethyl)-1H-pyrazol-5-ol

To a solution of 84 (100 mg, 0.17 mmol) in MeOH (8 mL) was added 37%formaldehyde (0.021 mL, 0.36 mmol) in an ice bath. The mixture wasstirred for 5 min. Sodium borohydride (32 mg, 0.84 mmol) was added. Thereaction mixture was stirred at 0° C. for 1 hour and concentrated todryness. The residue was purified by column chromatography (silica,0-80% CMA in CH₂Cl₂) to afford 85 (35 mg, 20%) as an off-white solid and86 (18 mg, 20%) as an off-white solid.

Data for 85:

R_(f) 0.35 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.15 (s, 3H), 1.21 (s, 6H), 1.35-2.35 (m, 20H), 2.35 (s, 6H),2.67 (m, 4H), 2.90 (m, 1H), 5.48 (s, 1H). mp 260-270° C. dec. APCI MS(Positive Mode) m/z 603 [C₃₇H₅₈N₆O+H]⁺.

Data for 86:

R_(f) 0.30 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.50 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 0.98(s, 3H), 1.15 (s, 3H), 1.21 (s, 6H), 1.35-2.08 (m, 20H), 2.30 (s, 2H),2.71 (m, 3H), 2.83 (m, 3H), 5.49 (s, 1H). mp 260-270° C. dec. APCI MS(Positive Mode) m/z 589 [C₃₆H₅₆N₆O+H]⁺.

Example 87

(i) Preparation of 87:1-(2-(3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-5-hydroxy-1H-pyrazol-4-yl)ethyl)urea

To a solution of 84 (66 mg, 0.11 mmol) and acetic acid (0.2 mL) in THF(2 mL) and benzene (3 mL) was added potassium cyanate (22 mg, 0.26mmol). The mixture was stirred at room temperature for 12 hours. Sodiumhydroxide (2 mL, 2.0 M) was added. The reaction mixture was stirred atroom temperature for 3 hours and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-10% MeOH in CMA) toafford the title compound (7 mg, 10%) as a brown solid.

R_(f) 0.50 (80:28:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.51 (s, 3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.00(s, 3H), 1.12 (s, 3H), 1.28 (s, 3H), 1.32 (s, 3H), 1.35-2.12 (m, 20H),2.28 (s, 1H), 2.30 (d, J=15.0 Hz, 1H), 2.60 (m, 2H), 2.85 (m, 1H), 5.52(s, 1H). mp>300° C. APCI MS (Positive Mode) m/z 618 [C₃₆H₅₅N₇O₂+H]⁺.

Example 88

(i) Preparation of 88b: Methyl3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-2-morpholino-3-oxopropanoate

To a solution of lithium bis(trimethylsilyl)amide (15.6 mL, 1 M in THF,15.6 mmol) was added methyl morpholinoacetate (1.8 g, 11.7 mmol) at −78°C. The mixture was stirred for 10 min. The anion solution was added to asolution of III (400 mg, 0.78 mmol) in THF (10 mL) precooled to −10° C.The mixture was stirred at −10° C. for 1 hour. The reaction mixture wasquenched with saturated NH₄Cl (60 mL) and extracted with EtOAc (100 mL).The organic phase was washed with brine, dried (MgSO₄), filtered andconcentrated to dryness. The residue was purified by columnchromatography (silica, 0-20% CMA in CH₂Cl₂) to afford the sub-titlecompound (120 mg) as a brown solid.

APCI MS (Positive Mode) m/z 635 [C₃₈H₅₈N₄O₄+H]⁺.

(ii) Preparation of 88:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-morpholino-1H-pyrazol-5-ol

A mixture of 88b (120 mg) and hydrazine (0.05 mL) in n-BuOH (2 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-30% CMA in CH₂Cl₂) to afford the titlecompound (6 mg, 5%) as a brown solid.

R_(f) 0.42 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.53 (s, 3H), 0.84 (s, 3H), 0.94 (s, 3H), 1.12(s, 3H), 1.21 (s, 3H), 1.23 (s, 6H), 1.32-2.37 (m, 24H), 2.53 (m, 1H),3.68 (m, 4H), 5.42 (s, 1H). mp>300° C. ESI MS (Positive Mode) m/z 617[C₃₇H₅₆N₆O₂+H]⁺.

Example 89

(i) Preparation of 89b: tert-Butyl 2-(4-methylpiperazin-1-yl)acetate

To a suspension of 1-methylpiperazine dihydrochloride (8.5 g, 49.2 mmol)and triethylamine (22.8 mL, 164.0 mmol) in THF (150 mL) was addedtert-butyl 2-bromoacetate (8.0 g, 41.0 mmol). The mixture was stirred atroom temperature for 12 hours. The reaction mixture was diluted withEtOAc (200 mL) and H₂O (100 mL). The organic phase was washed withbrine, dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-10% MeOH in CH₂Cl₂) toafford the sub-title compound (6.0 g, 69%).

¹H NMR (400 MHz, CDCl₃) δ 1.42 (s, 9H), 2.30 (s, 3H), 2.41-2.62 (m, 8H),3.10 (s, 2H).

(ii) Preparation of 89c: tert-Butyl3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-2-(4-methylpiperazin-1-yl)-3-oxopropanoate

To a solution of lithium bis(trimethylsilyl)amide (15.6 mL, 1 M in THF,15.6 mmol) was added 89b (2.5 g, 11.7 mmol) at −78° C. The mixture wasstirred for 10 min. The anion solution was added to a solution of III(400 mg, 0.78 mmol) in THF (10 mL) precooled to −10° C. The mixture wasstirred at 0° C. for 1.5 hours. The reaction mixture was quenched withsaturated NH₄Cl (60 mL) and extracted with EtOAc (100 mL). The organicphase was washed with brine, dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-20% CMA in CH₂Cl₂) to afford the sub-title compound (200 mg) as abrown solid.

APCI MS (Positive Mode) m/z 690 [C₄₂H₆₇N₅O₃+H]⁺.

(ii) Preparation of 89:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(4-methylpiperazin-1-yl)-1H-pyrazol-5-ol

A mixture of 89c (100 mg) and hydrazine (0.02 mL) in n-BuOH (2 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) to afford the titlecompound (16 mg, 18%) as a brown solid.

R_(f) 0.70 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.54 (s, 3H), 0.84 (s, 3H), 0.91 (s, 3H), 0.95(s, 3H), 1.01 (s, 3H), 1.10 (s, 3H), 1.11-2.06 (m, 16H), 2.20 (m, 1H),2.41 (m, 1H), 2.84 (s, 3H), 2.91-3.70 (m, 14H), 5.45 (s, 1H). mp>300° C.APCI MS (Positive Mode) m/z 630 [C₃₈H₅₉N₇O+H]⁺.

Example 90

(i) Preparation of 90b: tert-Butyl4-(2-tert-butoxy-2-oxoethyl)piperazine-1-carboxylate

To a suspension of tert-butyl piperazine-1-carboxylate (3.0 g, 16.1mmol) and triethylamine (4.5 mL, 32.2 mmol) in THF (80 mL) was addedtert-butyl 2-bromoacetate (2.4 mL, 16.1 mmol). The mixture was stirredat room temperature for 12 hours. The reaction mixture was diluted withEtOAc (200 mL) and H₂O (100 mL). The organic phase was washed withbrine, dried (MgSO₄), filtered and concentrated to dryness. The residuewas purified by column chromatography (silica, 0-5% MeOH in CH₂Cl₂) toafford the sub-title compound (4.2 g, 87%).

¹H NMR (400 MHz, CDCl₃) δ 1.42 (s, 18H), 2.50 (m, 4H), 3.10 (s, 2H),3.47 (m, 4H).

(ii) Preparation of 90c: tert-Butyl4-(1-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-3-tert-butoxy-1,3-dioxopropan-2-yl)piperazine-1-carboxylate

To a solution of lithium bis(trimethylsilyl)amide (19.4 mL, 1 M in THF,19.4 mmol) was added 90b (3.5 g, 11.7 mmol) at −78° C. The mixture wasstirred for 10 min. The anion solution was added to a solution of III(500 mg, 0.97 mmol) in THF (10 mL) precooled to −78° C. The mixture wasstirred at 0° C. for 1.5 hours. The reaction mixture was quenched withsaturated NH₄Cl (60 mL) and extracted with EtOAc (100 mL). The organicphase was washed brine, dried (MgSO₄), filtered and concentrated todryness. The residue was purified by column chromatography (silica,0-20% CMA in CH₂Cl₂) to afford the sub-title compound (237 mg) as abrown solid.

ESI MS (Positive Mode) m/z 776 [C₄₆H₇₃N₅O₅+H]⁺.

(iii) Preparation of 90d: tert-Butyl4-(3-((4aS,6aS,6bR,13aR)-12-amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-5-hydroxy-1H-pyrazol-4-yl)piperazine-1-carboxylate

A mixture of 90c (237 mg) and hydrazine (0.059 mL) in n-BuOH (5 mL) washeated at reflux overnight. The reaction mixture was concentrated todryness under reduced pressure. The residue was purified by columnchromatography (silica, 0-40% CMA in CH₂Cl₂) to afford the sub-titlecompound (15 mg, 7%) as a brown solid.

(iv) Preparation of 90:3-((4aS,6aS,6bR,13aR)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(piperazin-1-yl)-1H-pyrazol-5-ol

To a solution of 90d (15 mg) in MeOH (2 mL) was added HCl (0.8 mL, 2 Min diethyl ether) and stirred at room temperature for 5 hours. Thereaction mixture was concentrated and purified by preparative HPLC toprovide the title compound (4.5 mg, 30%) as an off-white solid.

R_(f) 0.12 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (400 MHz, CD₃OD) δ 0.55 (s, 3H), 0.86 (s, 3H), 0.89 (s, 3H), 0.91(s, 3H), 1.14 (s, 3H), 1.19 (s, 3H), 1.28 (s, 3H), 1.30-2.06 (m, 23H),2.20 (m, 1H), 2.41 (d, J=15.0 Hz, 1H), 2.85-3.65 (m, 4H), 5.45 (s, 1H).mp>300° C. APCI MS (Positive Mode) m/z 616 [C₃₇H₅₇N₇O+H]⁺.

Example 91

(i) Preparation of 91:(4aS,6aS,6bR,8aR,13aR,15bS)—S-5-Amino-4H-1,2,4-triazol-3-yl12-amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(pyridin-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbothioate

To a solution of 12 (150 mg, 0.26 mmol) in CH₂Cl₂ (10 mL) was addedthionyl chloride (0.19 mL, 2.6 mmol). The mixture was stirred at roomtemperature for 2 hours. The reaction mixture was concentrated todryness under reduced pressure. The residue was dissolved in pyridine(10 mL) and DMF (2 mL). 5-Amino-4H-1,2,4-triazole-3-thiol (71 mg, 0.60mmol) was added and stirred at room temperature for 1 hour. The reactionmixture was concentrated to dryness under reduced pressure. The residuewas purified by column chromatography (silica, 0-50% CMA in CH₂Cl₂)followed by preparative HPLC to afford the title compound (19 mg, 14%)as an off-white solid.

R_(f) 0.20 (80:18:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.30 (s, 3H), 0.82 (s, 3H), 0.90 (s, 3H), 0.98(s, 3H), 1.22 (s, 3H), 1.32 (s, 3H), 1.34 (s, 3H), 1.35-2.30 (m, 19H),2.35 (d, J=15.0 Hz, 1H), 2.75 (m, 1H), 7.91 (dd, J=5.7, 7.5 Hz, 1H),8.40 (d, J=7.8 Hz, 1H), 8.68 (d, J=4.8 Hz, 1H), 8.79 (s, 1H). mp>300° C.ESI MS (Positive Mode) m/z 669 [C₃₈H₅₂N₈OS+H]⁺.

Example 92

(i) Preparation of 92b:3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazole-4-a-carbonyl)-1-vinylpyrrolidin-2-one

To a solution of 32 (250 mg, 0.37 mmol) in CH₂Cl₂ (10 mL) was addedthionyl chloride (0.27 mL, 3.7 mmol). The mixture was stirred at roomtemperature for 2 hours. The reaction mixture was concentrated todryness under reduced pressure. The residue was dissolved in THF (5 mL)and added to a solution of 1-vinylpyrrolidin-2-one (0.39 mL, 3.7 mmol)and lithium bis(trimethylsilyl)amide (7.4 mL, 1 M in THF, 7.4 mmol) inTHF (5 mL) at −78° C. The reaction mixture was stirred at −78° C. for 1hour. The reaction mixture was quenched with saturated NH₄Cl (10 mL) andextracted with EtOAc (100 mL×2). The organic phase was washed with brinethen dried (MgSO₄), filtered and concentrated to dryness under reducedpressure. The residue was purified by column chromatography (silica,0-10% MeOH in CH₂Cl₂) to afford the sub-title compound (110 mg, 40%).

¹H NMR (300 MHz, CDCl₃) δ 0.51 (s, 3H), 0.81 (s, 3H), 0.87 (s, 6H), 1.10(s, 3H), 1.18 (s, 3H), 1.20 (s, 3H), 1.21-2.60 (m, 29H), 2.95 (m, 1H),3.35-3.75 (m, 8H), 4.05 (m, 1H), 4.45 (m, 2H), 6.40 (m, 1H), 7.01 (m,1H), 7.37 (m, 1H).

(ii) Preparation of 92:3-((4aS,6aS,6bR,8aR,13aR,15bS)-12-Amino-2,2,6a,6b,9,9,13a-heptamethyl-15-(2-(morpholinomethyl)furan-3-yl)-2,3,4,4a,5,6,6a,6b,7,8,8a,9,11,13,13a,13b,14,15b-octadecahydro-1H-chryseno[1,2-f]indazol-4-a-yl)-4-(2-aminoethyl)-1H-pyrazol-5-ol

A mixture of 92b (110 mg, 0.14 mmol) and hydrazine (0.5 mL) in EtOH (2mL) was sealed and heated to 160° C. by microwave for 3 hours. Thereaction mixture was concentrated to dryness. The residue was purifiedby column chromatography (silica, 0-70% CMA in CH₂Cl₂) to afford thetitle compound (25 mg, 25%).

R_(f) 0.54 (80:28:2 Methylene Chloride/Methanol/concentrated AmmoniumHydroxide).

¹H NMR (300 MHz, CD₃OD) δ 0.60 (s, 6H), 0.84 (s, 3H), 0.86 (s, 3H), 1.12(s, 3H), 1.22 (s, 3H), 1.28 (s, 3H), 1.35-2.0 (m, 19H), 2.18 (m, 2H),2.27 (d, J=15.0 Hz, 2H), 2.61-2.72 (m, 6H), 2.97 (m, 2H), 3.50-3.70 (m,7H), 6.41 (s, 1H), 7.51 (s, 1H). mp 238-240° C. APCI MS (Positive Mode)m/z 740 [C₄₄H₆₅N₇O₃+H]⁺.

Example IV

Table 6, below, lists the inhibitory concentrations of select compoundsand antibiotics in the biofilm growth assay against variousgram-negative bacterial biofilms. “N” means the number of isolates ofbacteria tested. The biofilm growth assay procedure detailed in ExampleI, above, was followed. Burkholderia cepacia used media consisting of M9salts, 100 μM CaCl2, 1 mM MgSO4, and 0.7% citrate in 0.4% Noble agar.Salmonella spp. used media consisting of Nutrient Broth with 0.5%glucose in 0.5% Noble agar. Compound 1 of the invention is the mostactive compound in Table 6. Compound 1 is superior to tobramycin,ceftazidime, and azithromycin in the biofilm growth assay.

A successful research lead optimization strategy requires examiningmultiple clinical isolates in parallel based upon the inherent risks ofantibacterial development and heterogeneity of biofilms. Past medicinalchemistry efforts focused on identifying superior analogs of approvedclasses of antibiotics have typically demonstrated that closely relatedanalogs can exhibit varying degrees of antibacterial activities againstdifferent isolates and species of bacteria in an unpredictable trend.Hence, accurately selecting the next set of synthetic targets requiresmicrobiological activities from a broad group of bacterial isolates andspecies of bacteria that would be encountered in clinical and communitysettings. In addition, significant variability of biofilm formationamong different clinical isolates of P. aeruginosa has been shown toexist. Therefore, the lead optimization of a biofilm inhibitor may bedetrimentally misguided if an unrepresentative group of clinicalisolates not exhibiting these different biofilms are used to generatestructure activity relationships.

TABLE 6 P. aeruginosa E. coli B. cepacia Salmonella Compound (n = 10) (n= 10) (n = 10) spp. (n = 6) Oleanolic Acid 8 16 0.5 >64 C644 2 >16 0.516 C649 16 >16 1 >16 Compound 1 1 1 0.25 1 Tobramycin 4 16 >64 8Ceftazidime 4 0.25 >8 0.5 Azithromycin >16 — >16 >16

Example V

The bioavailabilities of certain compounds were examined in mice.Administration of the compounds was performed orally and byintraperitoneal (IP) injection using a vehicle as known to those skilledin the art. Many vehicles can be used to examine bioavailability. Priorto administration, each vehicle was optimized based on compoundsolubility according to the formulation research conducted by Uckun etal. (Arznelmittel-Forschung (Drug Research) 2007; 57(4):218-226). Basedupon this publication, vehicles containing approximate ratios of 2:1:1of propylene glycol:Tween20:PEG400 (Tween20 being a common emulsier usedin formulations and food products) and less than 5% ethanol uponadministration exhibit good solubility properties and increase serumbioavailability. During these experiments, PEG400 demonstrated acritical role in serum bioavailability. The concentration of PEG400 ismodulated depending upon solubility of the compounds and the amount ofaqueous phase added (0.02 M citrate and 0.9% NaCl).

Compounds 1, 12, 64, and 68 demonstrated good bioavailability whenadministered orally or via IP injection at approximately 20 mg/kg to 50mg/kg exceeding approximately 5 μg/ml in the serum of mice at 30 minutesor 1 hour after administration. Based upon these results, many of thecompounds described herein would yield bioavailability including thecompounds in Example II, Tables 2 and 3. This example demonstrates thatthe compounds of the invention can be formulated into tablets, capsules,suppositories, and sterile liquids for parenteral administration asknown to those skilled in the art.

Example VI

The biofilm growth assay as described in Example I, as amended by thedetails that follow, was performed using Pseudomonas syringae,Xanthomonas campestris, and Pectobacterium atrosepticum (gram-negativebacterial plant pathogens). A freezer stock of each plant pathogen wasgrown separately overnight on 1.5% agar plates containing TSB at 30° C.The next day a pipette tip was used to inoculate a round plate composedof M8 salts and 0.7% glucose on 0.5% Noble agar from the overnight TSBplates. Plates were allowed to incubate at 30° C. for approximately 24to 48 hours. Compound 1 of the invention was examined against theseplant pathogens and found to inhibit the spreading biofilms at 0.03μg/ml against Pseudomonas syringae, 0.5 μg/ml against Xanthomonascampestris, and 0.06 μg/ml against Pectobacterium atrosepticum. Thesedata demonstrate that the compounds of the invention inhibit spreadingbiofilms of plant pathogens.

Example VII

INHIBITION OF THE GROWTH OF PREFORMED BIOFILMS. The Biofilm Growth Assaydescribed in Example I was performed in round plates with the compoundsof the invention only in agar on half of the plate. The agar notcontaining compound was inoculated with bacteria and the formed biofilmmoved toward the agar containing compound on the other half of the roundplate. Once the spreading biofilm reached the agar containing compound,the biofilm was inhibited from moving or spreading onto the agar withcompound. This Biofilm Growth Assay performed on these agar platesdemonstrates that the compounds of the invention inhibit the growth ofpreformed biofilms.

Approximately 0.5% agar not containing a compound of the invention waspoured and allowed to dry. Agar on half of the plate was removed andthen agar containing a compound of the invention was poured on the emptyhalf of the plate and allowed to dry. Bacteria is inoculated onto theagar not containing compound and allowed in incubate overnight asdescribed in Example I. During incubation bacteria spread as a biofilmuntil they reach the agar containing compound. The compounds of theinvention inhibit the spreading biofilm at the same concentrations atshown in Examples I, II, and III, above.

Example VIII

INHIBITION OF THE GROWTH OF PREFORMED BIOFILMS. The Biofilm Growth Assayas described in Example VII was performed in round plates with thecompounds of the invention in agar on half of the plate and antibioticdisks placed onto the agar containing the compounds of the invention.This assay demonstrates that the compounds of the invention aresynergistic with antibiotics like tobramycin and colistin at inhibitingspreading biofilms across the agar.

The Biofilm Growth Assay was performed as described in Example VIII. 2to 4 antibiotic disks were placed onto the agar containing a compound ofthe invention in a line parallel to the line that separates the twoagars in one round plate. Appropriate negative and positive controlplates were performed as known to those skilled in the art. This assaydemonstrates that the compounds of the invention in combination withantibiotics inhibit spreading biofilms at approximately 4 times less theconcentration than when performed alone as described in Example I.

Example IX Evaluation of Salts

Evaluation of Compound 62 (Table 4) salt formation was performed. HCl,HBr, H₃PO₄, p-toluenesulfonic acid, benzenesulfonic acid,methanesulfonic acid, L-tartaric acid, acetic acid, xinafoic acid,L-lactic acid, benzoic acid, adipic acid, oxalic acid, pamoic acid,maleic acid, and laurylsulfuric acid indicated crystallinity.p-toluenesulfonic acid (mono) and H₃PO₄ (bis) provided the mostdesirable physical properties compared to freebase. As stated herein,the invention also includes compounds and their salts.

Evaluation of Compound 64 (Table 4) salt formation was performed. HCl(mono and bis), p-TSA (bis), BSA (bis), MSA (bis), H₂SO₄ (mono), andH₃PO₄ (mono) provided salts with crystallinity. Bis-MSA and mono-H₃PO₄increased aqueous solubility compared to freebase. As stated herein, theinvention also includes compounds and their salts.

Example X

A topical gel containing 2% by weight of the compound of the inventionwith azithromycin for use in treating skin infections can be prepared.

0.25 gram of the compound of the invention is dissolved in 6.75 grams ofethanol. 0.2 grams of azithromycin is dissolved in this solution. 0.25grams of hydroxypropyl methylcellulose is added with gentle stiflinguntil a homogenous solution is obtained. 4.8 grams of water is thenadded with gentle shaking.

A formulation without antibiotic can also be prepared using this sameprocedure.

Example XI Pharmaceutical Formulation for Nebulization of a Compound ofthe Invention

Solutions were prepared comprising 2 mg/ml and 10 mg/ml of the compoundof the invention in ethanol/propylene glycol/water (85:10:5). Thesesolutions were nebulized separately by a ProNeb Ultra nebulizermanufactured by PARI. The nebulized solutions were collected in a coldtrap, processed appropriately, and were detected by mass spectrometry.The compound of the invention is expected to be recovered from bothformulations to demonstrate that nebulization can be used to deliverthis compound to patients with lung infections.

Example XII

The compounds shown in the table below were prepared semi-syntheticallyand tested in the biofilm growth assay as described herein. Thesecompounds with substitutions at R¹ and R² did not inhibit the biofilmsof P. aeruginosa or E. coli at 1 or 2 μg/ml sufficiently to warrantfurther investigation relative to the compounds noted above.

  R¹

  R²

All references, including without limitation all papers, publications,presentations, texts, reports, manuscripts, brochures, internetpostings, journal articles, periodicals, and the like, cited in thisspecification are hereby incorporated by reference. The discussion ofthe references herein is intended merely to summarize the assertionsmade by their authors and no admission is made that any referenceconstitutes prior art. The inventors reserve the right to challenge theaccuracy and pertinence of the cited references.

It is intended that all patentable subject matter disclosed herein beclaimed and that no such patentable subject matter be dedicated to thepublic. Thus, it is intended that the claims be read broadly in light ofthat intent. In addition, unless it is otherwise clear to the contraryfrom the context, it is intended that all references to “a” and “an” andsubsequent corresponding references to “the” referring back to theantecedent basis denoted by “a” or “an” are to be read broadly in thesense of “at least one.” Similarly, unless it is otherwise clear to thecontrary from the context, the word “or,” when used with respect toalternative named elements is intended to be read broadly to mean, inthe alternative, any one of the named elements, any subset of the namedelements or all of the named elements.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results obtained. Itshould be understood that the aforementioned embodiments are forexemplary purposes only and are merely illustrative of the many possiblespecific embodiments that can represent applications of the principlesof the invention. Thus, as various changes could be made in the abovemethods and compositions without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription as shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

Moreover, one of ordinary skill in the art can make various changes andmodifications to the invention to adapt it to various usages andconditions, including those not specifically laid out herein, withoutdeparting from the spirit and scope of this invention. Accordingly,those changes and modifications are properly, equitably, and intended tobe, within the full range of equivalents of the invention disclosed anddescribed herein.

What is claimed is: 1-62. (canceled)
 63. A compound corresponding to thefollowing chemical structure:

wherein R1 is selected from the group consisting of hydrogen, methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, lower cycloalkyl, lowercycloalkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; wherein R² is selected from the group consisting ofcarboxyl, amide, hydroxyamide, methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

wherein R³ is selected from the group consisting of hydrogen and methyl;wherein one of R⁴ and R⁵ is hydrogen and the other is methyl; wherein R⁶and R⁷ are independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, isopropyl, and butyl; wherein R⁸ is selected fromthe group consisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; whereinR⁹ is selected from the group consisting of hydrogen, halide, loweralkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl, loweralkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lowercycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyloptionally substituted with moieties selected from the group consistingof hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, loweralkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, carboxyl,amide, hydroxyamide, —CONHCH₃, —NHCONH₂, —SO₂NH₂, —SO₂CH₃, —NHCOCH₃,—NHCSNH₂, and —NHSO₂CH₃; and wherein R¹⁰ and R¹¹ together with thecarbon atoms to which they are attached form an aromatic N-heterocyclewhich is substituted or not selected from the group consisting ofindole, imidazole, quinoline, isoquinoline, pyridine, triazine,pyrazine, pyrimidine, pyridazine, isoindole, pyrrole, benzimidazole,purine, pyrazole, benzopyrazole, indazole, quinoxaline, acridine,quinazoline, indolizine, carbazole and cinnoline; and salts thereof. 64.A compound according to claim 1, wherein the compound corresponds to thefollowing chemical structure:

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, lower cycloalkyl, lowercycloalkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; wherein R² is selected from the group consisting ofcarboxyl, amide, hydroxyamide, methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

wherein R³ is selected from the group consisting of hydrogen and methyl;wherein one of R⁴ and R⁵ is hydrogen and the other is methyl; wherein R⁶and R⁷ are independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, isopropyl, and butyl; wherein R⁸ is selected fromthe group consisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; andwherein R⁹ is selected from the group consisting of hydrogen, halide,lower alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lowercycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyloptionally substituted with moieties selected from the group consistingof hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, loweralkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, carboxyl,amide, hydroxyamide, —CONHCH₃, —NHCONH₂, —SO₂NH₂, —SO₂CH₃, —NHCOCH₃,—NHCSNH₂, and —NHSO₂CH₃; and salts thereof.
 65. A compound according toclaim 63 wherein R¹ is selected from the group consisting of methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, lower cycloalkyl, lowercycloalkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl, preferably R¹ is selected from the group consisting ofpyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl,thiazolyl, isothiazolyl, thienyl, furanyl, furazanyl, pyridinyl,pyrimidinyl, pyridazinyl, indolyl, 3H-indolyl, isoindolyl, indolinyl,indolizinyl, indazolyl, dihydroindolyl, tetrahydroindolyl, purinyl,pyrazinyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, benzimidazolyl,benzopyranyl, benzoxazolyl, benzisoxazolyl, benzofuranyl,isobenzofuranyl, benzothiazolyl, benzisothiazolyl, benzothienyl,furopyridinyl, phthalazinyl, napthyridinyl, pyrazolopyridyl,pyrazolopyrimidinyl, pyrrolopyridinyl, and tetrahydropyrrolopyridinyloptionally substituted from moieties selected from the group consistingof lower alkyl, halide, lower haloalkyl, hydroxyl, amino, loweraminoalkyl, lower hydroxyalkyl, nitrile, lower alkyl nitrile, loweralkyl ethers, lower alkoxy, lower alkoxyalkyl, lower alkoxycarbonyl,lower alkylcarbonylamino, and thioalkyl.
 66. A compound according toclaim 64 wherein the compound corresponds to the following chemicalstructure:

wherein R¹ is selected from the group consisting of hydrogen, halide,lower alkyl, substituted lower alkyl, lower alkenyl, substituted loweralkenyl, lower cycloalkyl, lower cycloalkenyl, aryl, substituted aryl,heteroaryl, and substituted heteroaryl, preferably R¹ is hydrogen.
 67. Acompound according to claim 66 wherein R¹ is selected from the groupconsisting of halide, lower alkyl, substituted lower alkyl, loweralkenyl, substituted lower alkenyl, lower cycloalkenyl, aryl,substituted aryl, heteroaryl, and substituted heteroaryl, preferably R¹is selected from the group consisting of pyrrolyl, pyrazolyl,imidazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thienyl, furanyl, furazanyl, pyridinyl, pyrimidinyl, pyridazinyl,indolyl, 3H-indolyl, isoindolyl, indolinyl, indolizinyl, indazolyl,dihydroindolyl, tetrahydroindolyl, purinyl, pyrazinyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, benzimidazolyl, benzopyranyl, benzoxazolyl,benzisoxazolyl, benzofuranyl, isobenzofuranyl, benzothiazolyl,benzisothiazolyl, benzothienyl, furopyridinyl, phthalazinyl,napthyridinyl, pyrazolopyridyl, pyrazolopyrimidinyl, pyrrolopyridinyl,and tetrahydropyrrolopyridinyl optionally substituted from moietiesselected from the group consisting of lower alkyl, halide, lowerhaloalkyl, hydroxyl, amino, lower aminoalkyl, lower hydroxyalkyl,nitrile, lower alkyl nitrile, lower alkyl ethers, lower alkoxy, loweralkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, andthioalkyl.
 68. A compound according to claim 64 wherein R¹ is selectedfrom the group consisting of hydrogen, halide, lower alkyl, substitutedlower alkyl, lower alkenyl, substituted lower alkenyl, substituted aryl,lower cycloalkenyl, aryl, heteroaryl, and substituted heteroaryl; andwherein R² is selected from the group consisting of amide, hydroxyamide,methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,


69. A compound according to claim 68 wherein R¹ is selected from thegroup consisting of pyrrolyl, pyrazolyl, imidazolyl, oxazolyl,isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thienyl, furanyl,furazanyl, pyridinyl, pyrimidinyl, pyridazinyl, indolyl, 3H-indolyl,isoindolyl, indolinyl, indolizinyl, indazolyl, dihydroindolyl, andtetrahydroindolyl optionally substituted from moieties selected from thegroup consisting of lower alkyl, halide, lower haloalkyl, hydroxyl,amino, lower aminoalkyl, lower hydroxyalkyl, nitrile, lower alkylnitrile, lower alkyl ethers, lower alkoxy, lower alkoxyalkyl, loweralkoxycarbonyl, lower alkylcarbonylamino, and thioalkyl.
 70. A compoundaccording to claim 64 wherein R² is

wherein R⁸ is selected from the group consisting of hydroxyl and amino;and wherein R⁹ is selected from the group consisting of hydrogen,halide, lower alkyl, lower alkenyl, lower alkynyl, morpholinyl,piperazinyl, lower alkyl piperazinyl, heterocycloalkyl, lowercycloalkyl, lower cycloalkenyl; and lower alkyl, lower alkenyl, andlower alkynyl optionally substituted with moieties selected from thegroup consisting of hydroxyl, amino, lower aminoalkyl, halide, loweralkoxy, lower alkoxyalkyl, lower alkoxycarbonyl, loweralkylcarbonylamino, carboxyl, amide, hydroxyamide, —CONHCH₃, —NHCONH₂,—SO₂NH₂, —SO₂CH₃, —NHCOCH₃, —NHCSNH₂, and —NHSO₂CH₃ preferably R⁹ ishydrogen; and salts thereof.
 71. A compound according to claim 70wherein R¹ is selected from the group consisting of heteroaryl andsubstituted heteroaryl, wherein R² is

wherein R⁸ is selected from the group consisting of hydroxyl and amino;and wherein R⁹ is hydrogen.
 72. A compound according to claim 64 whereinthe compound corresponds to the following chemical structure:

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, lower alkyl, substituted lower alkyl, lower alkenyl, substitutedlower alkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl, preferably R¹ is selected from the group consisting ofheteroaryl and substituted heteroaryl.
 73. A compound according to claim63, wherein the compound corresponds to the following chemicalstructure:

wherein R1 is selected from the group consisting of hydrogen, methyl,halide, nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; whereinR3 is selected from the group consisting of hydrogen and methyl; andwherein one of R4 and R5 is hydrogen and the other is methyl; and saltsthereof.
 74. A compound according to claim 63 wherein the compoundcorresponds to the following chemical structure:


75. A method for inhibiting or reducing the formation or growth of abiofilm comprising contacting said biofilm or a cell capable of biofilmformation or growth with an effective amount of a compound or acomposition comprising the compound corresponding to the followingchemical structure:

wherein R1 is selected from the group consisting of hydrogen, methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, lower cycloalkyl, lowercycloalkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; wherein R² is selected from the group consisting ofcarboxyl, amide, hydroxyamide, methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

wherein R³ is selected from the group consisting of hydrogen and methyl;wherein one of R⁴ and R⁵ is hydrogen and the other is methyl; wherein R⁶and R⁷ are independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, isopropyl, and butyl; wherein R⁸ is selected fromthe group consisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; whereinR⁹ is selected from the group consisting of hydrogen, halide, loweralkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl, loweralkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lowercycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyloptionally substituted with moieties selected from the group consistingof hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, loweralkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, carboxyl,amide, hydroxyamide, —CONHCH₃, —NHCONH₂, —SO₂NH₂, —SO₂CH₃, —NHCOCH₃,—NHCSNH₂, and —NHSO₂CH₃; and wherein R¹⁰ and R¹¹ together with thecarbon atoms to which they are attached form an aromatic N-heterocyclewhich is substituted or not selected from the group consisting ofindole, imidazole, quinoline, isoquinoline, pyridine, triazine,pyrazine, pyrimidine, pyridazine, isoindole, pyrrole, benzimidazole,purine, pyrazole, benzopyrazole, indazole, quinoxaline, acridine,quinazoline, indolizine, carbazole and cinnoline; and salts thereof. 76.A method according to claim 75 wherein the compound corresponds to thefollowing chemical structure:

wherein R¹ is selected from the group consisting of hydrogen, methyl,halide, lower haloalkyl, nitrile, lower alkyl nitrile, lower alkyl,substituted lower alkyl, lower alkenyl, substituted lower alkenyl, loweralkynyl, substituted lower alkynyl, lower cycloalkyl, lowercycloalkenyl, aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; wherein R² is selected from the group consisting ofcarboxyl, amide, hydroxyamide, methylamide, —CH₂N(CH₃)₂, —CH₂NR⁶R⁷,

wherein R³ is selected from the group consisting of hydrogen and methyl;wherein one of R⁴ and R⁵ is hydrogen and the other is methyl; wherein R⁶and R⁷ are independently selected from the group consisting of hydrogen,methyl, ethyl, propyl, isopropyl, and butyl; wherein R⁸ is selected fromthe group consisting of hydroxyl, amino, —N(CH₃)₂, and —NHCH₃; andwherein R⁹ is selected from the group consisting of hydrogen, halide,lower alkyl, lower alkenyl, lower alkynyl, morpholinyl, piperazinyl,lower alkyl piperazinyl, heterocycloalkyl, lower cycloalkyl, lowercycloalkenyl; and lower alkyl, lower alkenyl, and lower alkynyloptionally substituted with moieties selected from the group consistingof hydroxyl, amino, lower aminoalkyl, halide, lower alkoxy, loweralkoxyalkyl, lower alkoxycarbonyl, lower alkylcarbonylamino, carboxyl,amide, hydroxyamide, —CONHCH₃, —NHCONH₂, —SO₂NH₂, —SO₂CH₃, —NHCOCH₃,—NHCSNH₂, and —NHSO₂CH₃; and salts thereof.
 77. A method according toclaim 75 wherein the compound corresponds to the following chemicalstructure:

wherein R1 is selected from the group consisting of hydrogen, methyl,halide, nitrile, lower alkyl, substituted lower alkyl, lower alkenyl,substituted lower alkenyl, lower alkynyl, substituted lower alkynyl,aryl, substituted aryl, heteroaryl, and substituted heteroaryl; whereinR3 is selected from the group consisting of hydrogen and methyl; andwherein one of R4 and R5 is hydrogen and the other is methyl; and saltsthereof.
 78. A method according to claim 75, wherein the compoundcorresponds to the followings chemical structure: